Method of treating airway diseases with β-adrenergic inverse agonists

ABSTRACT

The use of β-adrenergic inverse agonists provides a new and highly efficient way of treating a number of pulmonary airway diseases, including asthma, emphysema, and chronic obstructive pulmonary diseases. In general, such a method comprises administering a therapeutically effective amount of a β-adrenergic inverse agonist to the subject to treat the pulmonary airway disease. Particularly preferred inverse agonists include nadolol and carvedilol.

CROSS-REFERENCES

This application claims priority from Provisional Application Ser. No.60/510,250, by Richard A. Bond, entitled “Method for Treating AirwayDiseases with Beta-Adrenergic Inverse Agonists,” filed Oct. 9, 2003,which is incorporated herein in its entirety by this reference.

STATEMENT REGARDING FEDERAL FUNDED RESEARCH

Certain of the research leading to the invention recited in thisapplication has been funded by grants from the National Institutes ofHealth. The United States government may therefore have certain rightsin this invention.

BACKGROUND OF THE INVENTION

The present invention relates to novel methods for preventing, treating,or reducing the severity of diseases and conditions mediated byβ-adrenergic receptors, particularly pulmonary airway diseases. Inparticular, it provides for methods and compositions for treatingpulmonary airway diseases by long-term administration of β-adrenergicinverse agonist drugs, either alone or in combination with other drugs,such as β₂-agonists, steroids, leukotriene modifiers, anticholinergics,methylxanthines, phosphodiesterase-4 inhibitors, or anti-IgE antibodies.

Many diseases and conditions are mediated by β-adrenergic receptors. Inparticular, these receptors are involved in many pulmonary airwaydiseases. Pulmonary airway diseases are characterized by reducedpulmonary function and airway flow. These symptoms are often due tosecretion of mucus or tissue damage. These diseases include allergicrhinitis (“hay fever”), asthma, cystic fibrosis, chronic obstructivepulmonary disease (COPD), Churg-Strauss syndrome, bronchitis,bronchiectasis, and emphysema. These diseases are serious and areresponsible for significant mortality and morbidity.

COPD patients have obstructed airflow in the lungs. There are a numberof ways that patients develop COPD. However, the hallmark of thedisorder is dyspnea, or breathlessness. COPD is frequently associatedwith long-term cigarette smoking and can develop as the result ofuntreated allergic conditions. The aging process can also cause thebronchi and bronchioles to lose their elasticity.

Churg-Strauss syndrome is an inflammatory disease in which patientsexhibit asthmatic symptoms such as airway hyperreactivity. Inflammationof pulmonary airways occurs, compromising pulmonary function.

In bronchitis, airway function is compromised due to hypersecretion ofmucus, initially due to irritants. Bronchitis can be the result ofinfection or allergic reaction. With chronic bronchitis, coughing ispersistent but may no longer be sufficient to clear airways, leading toairflow obstruction. Chronic bronchitis affects the bronchial tubes.

Bronchiectasis results from infection in the lungs, leading toirreversible airway damage. Patients often complain of persistent coughand expectorate a foul-smelling sputum. The consequences of theinfection, in conjunction with the secretions, contributes to airwayobstruction despite the fact that bronchi and bronchioles can beexceptionally dilated.

Patients with emphysema have reduced pulmonary function due todestructive damage of the walls of lung alveoli. Often, patients arelong-time smokers and have elevated levels of inflammatory cells, suchas neutrophils and macrophages, in the lungs; other pathophysiologicprocesses are at work as well. The smoke is believed to activate lungneutrophils to release elastase, a damaging proteolytic enzyme. Otherenvironmental irritants can also be involved in emphysema.

Asthma alone is a chronic problem for 20 million American patients. Therate of occurrence of asthma has been increasing rapidly in the UnitedStates, particularly in urban areas, and particularly in children. Thecause of this increase is not known, but exposure to environmentalpollutants is suspected. The age-adjusted mortality rate for asthma inthe United States increased 55.6% between 1979 and 1998 (American LungAssociation's Epidemiology and Statistics Unit, Best Practices andProgram Services. Trends in Asthma Morbidity and Mortality, 2002).Persons suffering from asthma are often sensitive to allergens, such ashousehold dust, animal dander, and pollen (allergic asthma). However,intrinsic asthma can be triggered in a patient by emotional distress orpanic, as well as by factors such as exposure to cold or exercise, or byadministration of certain medications such as aspirin. In asthma,patients exhibit airway hyperresponsiveness to these provocations. Thesetrigger immune system cells to release histamines, IgE molecules,cytokines, or chemokines. Airway smooth muscle responds acutely to theseprovocations, resulting in bronchial constriction. Additionally, theairway becomes damaged and inflamed, and mucus is secreted, furtherlimiting airway flow. Asthma attacks are characterized by shortness ofbreath, caused by contraction of the smaller bronchi and bronchioles,chest tightness, coughing, and wheezing. The attacks can be mild,moderate, or severe.

Patients with these airway disorders may have airway spasms, furtherreducing airflow through the pulmonary tree. During an attack, apatient's airway is constricted, leading to difficulty breathing. Airwaysmooth muscle is responsible for the bronchoconstriction. The airwaysmooth muscle cells express β₂-adrenergic receptors. Agonist binding tothese receptors, such as by epinephrine or other β₂-agonist drugs,results in smooth muscle relaxation.

Consequently, for acute bronchospasms many patients inhale short-actingβ₂-adrenergic agonists to prevent or reduce the severity of asthmaattacks.

However, chronic administration of β₂-adrenergic agonists has beendemonstrated to lead to drug tolerance and reduced therapeutic effect ontheir continued administration. Reduced responsiveness, also known astachyphylaxis or tolerance, results from a culmination of events, whichinclude desensitization, sequestration, and down-regulation ofreceptors. Furthermore, there is also an increased hyperresponsivenessof the pulmonary airway in response to provocations such as allergens.

Epidemiological studies have demonstrated a positive correlation betweenthe chronic use of short-acting β₂-adrenergic agonists and asthmamortality. A large trial with the long-acting β₂-adrenergic agonist,salmeterol, was stopped due to increased death rates. This underscoresthat, while short-term administration of β₂-adrenergic agonists may behelpful to asthmatic patients and to patients with other diseases andconditions modulated by β₂-adrenergic receptors, long-termadministration of these agonists may be deleterious.

Conventional wisdom in the management of asthma and other diseases andconditions in which airway hyperresponsiveness and bronchoconstrictionoccur is that the administration of beta blockers, such as those thatare frequently used in the treatment of cardiovascular conditions, aredefinitely contraindicated for asthmatic patients. In T. Clark & J.Rees, “Practical Management of Asthma” (2d ed, Martin Dunitz, 1996), itstates: “These (β-blockers) often produce adverse effects when given toasthmatics, Treatment with beta blockers can also bring to lightpreviously undiagnosed asthma. Fatal bronchoconstriction has beenproduced by a single dose of beta blockers. It is best to avoid all betablockers in asthmatics.”

Therefore, there needs to be increased focus on the bronchoconstrictionoccurring in asthma. This can progress to status asthmaticus. Moreefficient and long-lasting therapeutic modalities that can reverse thebronchoconstriction and can bring about dilation of the airways areneeded.

Consequently, there is a tremendous need for new therapeuticalternatives to β₂-adrenergic agonist use in asthmatics and in patientssuffering from other diseases and conditions modulated by β₂-adrenergicreceptors, particularly diseases affecting the respiratory system suchas asthma.

SUMMARY OF THE INVENTION

One embodiment of the invention is a method for treatment of pulmonaryairway disease in a subject suffering from pulmonary airway diseasecomprising administering a therapeutically effective amount of aβ-adrenergic inverse agonist to the subject to treat the pulmonaryairway disease.

Particularly preferred inverse agonists are nadolol, carvedilol,metoprolol, timolol, and ICI 118,551.

The pulmonary airway disease can be selected from the group consistingof asthma, bronchiectasis, bronchitis, chronic obstructive pulmonarydisease, Churg-Strauss syndrome, the pulmonary sequelae of cysticfibrosis, emphysema, allergic rhinitis, and pneumonia.

In this method, the β-adrenergic inverse agonist can be administeredover time in a series of graduated doses starting with the lowest doseand increasing to the highest dose.

Another aspect of the invention is a pharmaceutical compositioncomprising:

(1) nadolol in a quantity selected from the group consisting of 1 mg, 3mg, 5, mg, 10 mg, 15 mg, 30 mg, 50 mg, and 70 mg; and

(2) a pharmaceutically acceptable carrier.

Yet another aspect of the invention is a blister pack comprising:

(1) a lower substrate;

(2) an intermediate dosage holder that is shaped to generate a pluralityof cavities and that is placed over the lower substrate, the cavitiesbeing shaped to hold dosage forms of a β-adrenergic inverse agonist;

(3) an upper substrate placed over the intermediate dosage holder thathas a plurality of apertures, each aperture being located to accommodatea corresponding cavity; wherein the dosage forms are of graduateddosages starting with a lowest dose and proceeding to a highest dose;and

(4) dosage forms of a β-adrenergic inverse agonist placed in thecavities.

Yet another aspect of the invention is a blister pack comprising:

(1) a lower substrate;

(2) an intermediate dosage holder that is shaped to generate a pluralityof cavities, the cavities being shaped to hold dosage forms of aβ-adrenergic inverse agonist;

(3) an upper substrate placed over the intermediate dosage holder thathas a plurality of apertures, each aperture being located to accommodatea corresponding cavity; and

(4) dosage forms of a β-adrenergic inverse agonist placed in thecavities, wherein the dosage forms are of at least two dosages of aβ-adrenergic inverse agonist: (i) a maintenance dose that is the highestdose in a series of graduated doses; and (ii) at least one backuprestoration dose or a lower dose to be taken in a specified condition.

Still another aspect of the invention is a method for treatment ofpulmonary airway disease in a subject suffering from pulmonary airwaydisease comprising administering to the subject: (1) a therapeuticallyeffective amount of a β-adrenergic inverse agonist and (2) atherapeutically effective amount of a β₂-selective adrenergic agonist inorder to treat the pulmonary airway disease.

Still another aspect of the invention is a method for treatment ofpulmonary airway disease in a subject suffering from pulmonary airwaydisease comprising administering to the subject: (1) a therapeuticallyeffective amount of a β-adrenergic inverse agonist and (2) atherapeutically effective amount of a steroid in order to treat thepulmonary disease.

Still another aspect of the invention is a method for treatment ofpulmonary airway disease in a subject suffering from pulmonary airwaydisease comprising administering to the subject: (1) a therapeuticallyeffective amount of a β-adrenergic inverse agonist and (2) atherapeutically effective amount of an anticholinergic drug in order totreat the pulmonary airway disease.

Still another aspect of the invention is a method for treatment ofpulmonary airway disease in a subject suffering from pulmonary airwaydisease comprising administering to the subject: (1) a therapeuticallyeffective amount of a β-adrenergic inverse agonist and (2) atherapeutically effective amount of a xanthine compound in order totreat the pulmonary airway disease.

Still another aspect of the invention is a method for treatment ofpulmonary airway disease in a subject suffering from pulmonary airwaydisease comprising administering to the subject: (1) a therapeuticallyeffective amount of a β-adrenergic inverse agonist and (2) atherapeutically effective amount of an anti-IgE antibody in order totreat the pulmonary airway disease.

Still another aspect of the invention is a method for treatment ofpulmonary airway disease in a subject suffering from pulmonary airwaydisease comprising administering to the subject: (1) a therapeuticallyeffective amount of a β-adrenergic inverse agonist and (2) atherapeutically effective amount of a leukotriene modifier in order totreat the pulmonary airway disease.

Still another aspect of the invention is a method for treatment ofpulmonary airway disease in a subject suffering from pulmonary airwaydisease comprising administering to the subject: (1) a therapeuticallyeffective amount of a β-adrenergic inverse agonist and (2) atherapeutically effective amount of phosphodiesterase IV inhibitor inorder to treat the pulmonary airway disease.

Still another aspect of the invention is a pharmaceutical compositioncomprising:

(1) a therapeutically effective amount of a β-adrenergic inverseagonist;

(2) a therapeutically effective amount of a second therapeutic agenteffective to treat a pulmonary airway disease, the second therapeuticagent being selected from the group consisting of a β₂-selectiveadrenergic agonist, a steroid, an anticholinergic drug, a xanthinecompound, an anti-IgE antibody, a leukotriene modifier, and aphosphodiesterase IV inhibitor; and

(3) a pharmaceutically acceptable carrier.

Still another aspect of the invention is a blister pack comprising:

(1) a lower substrate;

(2) an intermediate dosage holder that is shaped to generate a pluralityof cavities and that is placed over the lower substrate, the cavitiesbeing shaped to hold dosage forms of the pharmaceutical compositionincluding both a therapeutic amount of a β-adrenergic inverse agonistand a therapeutic amount of a second therapeutic agent effective totreat a pulmonary airway disease;

(3) an upper substrate placed over the intermediate dosage holder thathas a plurality of apertures, each aperture being located to accommodatea corresponding cavity; and

(4) dosage forms of the pharmaceutical composition placed in thecavities.

Yet another aspect of the invention is a blister pack comprising:

(1) a lower substrate;

(2) an intermediate dosage holder that is shaped to generate a pluralityof cavities and that is placed over the lower substrate, the cavitiesbeing shaped to hold dosage forms of: (a) a first pharmaceuticalcomposition that comprises: (i) a therapeutically effective amount of aβ-adrenergic inverse agonist; and (ii) a first pharmaceuticallyacceptable carrier; and (b) a second pharmaceutical composition thatcomprises: (i) a therapeutically effective amount of a secondtherapeutic agent effective to treat a pulmonary airway disease, thesecond therapeutic agent being selected from the group consisting of aβ₂-selective adrenergic agonist, a steroid, an anticholinergic drug, axanthine compound, an anti-IgE antibody, a leukotriene modifier, and aphosphodiesterase IV inhibitor; and (ii) a second pharmaceuticallyacceptable carrier;

(3) an upper substrate placed over the intermediate dosage holder thathas a plurality of apertures, each aperture being located to accommodatea corresponding cavity; and

(4) dosage forms of the first and second pharmaceutical compositionsplaced in the cavities.

BRIEF DESCRIPTION OF THE DRAWINGS

The following invention will become better understood with reference tothe specification, appended claims, and accompanying drawings, where:

FIG. 1 is a diagram of a blister pack holding dosage forms of inverseagonists according to the invention.

FIG. 2A is a graph showing that methacholine provocation significantlyenhances airway resistance (R_(aw)) in asthmatic mice.

FIG. 2B is a similar graph showing that saline provocation, as acontrol, does not significantly enhance airway resistance in asthmaticmice.

FIG. 2C is a similar graph showing that the administration of a singleintravenous bolus of salbutamol to asthmatic mice reduced the level ofairway responsiveness to methacholine provocation and the level ofairway resistance.

FIG. 2D is a similar graph showing that no protection was observed whensalbutamol was delivered to the mice for 28 days before methacholineprovocation.

FIG. 2E is a similar graph showing that when asthmatic mice were given asingle intravenous bolus of alprenolol, a β-adrenergic antagonist withpartial agonist activity, their airway responsiveness was diminished.

FIG. 2F is a similar graph showing that when asthmatic mice were exposedto alprenolol for 28 days, their average methacholine dose-responserelationship was similar to that obtained in nontreated mice,demonstrating that this drug provides no benefit upon chronicadministration.

FIG. 2G is a similar graph showing that a single intravenous bolus ofcarvedilol enhanced the airway responsiveness in the asthmatic mice.

FIG. 2H is a similar graph showing that chronic administration ofcarvedilol reduced the responsiveness of asthmatic mice to methacholineprovocation.

FIG. 2I is a similar graph showing that a single intravenous bolus ofnadolol also enhanced the airway responsiveness of asthmatic micesimilar to that observed for carvedilol.

FIG. 2J is a similar graph showing that chronic administration ofnadolol reduced the responsiveness of asthmatic mice to methacholineprovocation, again, similar to that observed for carvedilol.

FIG. 3 is a graph showing the effects of administration of β-adrenergicreceptor ligands on the peak airway responsiveness to cholinergicstimulation ((A), after treatments with the β-adrenergic agonistsalbutamol; (B), after acute treatments with β-adrenergic receptorinverse agonists; and (C) after chronic treatment with β-adrenergicreceptor inverse agonists.

FIG. 4 is a series of epifluorescent photomicrographs showing anincrease in β-adrenergic receptor density upon treatment with nadolol.

FIG. 5A is a graph showing the effect of combination therapy withcarvedilol and salbutamol on airway hyperresponsiveness in asthmaticmice challenged with methacholine.

FIG. 5B is a summary graph showing the results presented in FIG. 5A.

FIG. 6 is a graph showing the effect of acute combination therapy withnadolol and aminophylline on airway hyperresponsiveness in asthmaticmice challenged with methacholine.

FIG. 7 is a graph showing the ratio of phospholipase C to actin in micetreated with various treatments, including long-term nadololadministration, to show that long-term nadolol administration decreasesthe activity of phospholipase C.

FIG. 8A is a graph showing the effects of salbutamol upon airwayhyperresponsiveness.

FIG. 8B is a graph showing the effects of high-dose alprenolol uponairway hyperresponsiveness.

FIG. 8C is a graph showing the effects of low-dose alprenolol uponairway hyperresponsiveness.

FIG. 8D is a graph showing the effects of high-dose carvedilol uponairway hyperresponsiveness.

FIG. 8E is a graph showing the effects of low-dose carvedilol uponairway hyperresponsiveness.

FIG. 8F is a graph showing the effects of high-dose nadolol upon airwayhyperresponsiveness.

FIG. 8G is a graph showing the effects of low-dose nadolol upon airwayhyperresponsiveness.

FIG. 9 is a set of graphs showing the effects of long-term dosage ofmetoprolol and timolol upon airway hyperresponsiveness in asthmaticmice: (A) experimental results with metoprolol and timolol; (B)historical controls with non-challenged mice (Ctrl) and with challengedmice with no treatment (NTX).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, in the generally accepted two-state model of receptortheory, the term “agonist” is defined as a substance that has anaffinity for the active site of a receptor and thereby preferentiallystabilizes the active state of the receptor, or a substance, including,but not limited to, drugs, hormones, or neurotransmitters, that producesactivation of receptors and enhances signaling by those receptors.Irrespective of the mechanism or mechanisms of action, an agonistproduces activation of receptors and enhances signaling by thosereceptors.

As used herein, in the two-state model of receptor theory, the term“antagonist” is defined as a substance that does not preferentiallystabilize either form of the receptor, active, or inactive, or asubstance, including, but not limited to, drugs, hormones, andneurotransmitters, that prevents or hinders the effects of agonistsand/or inverse agonists. Irrespective of the mechanism or mechanisms ofaction, an antagonist prevents or hinders the effects of agonists and/orinverse agonists.

As used herein, in the two-state model of receptor theory, the term“inverse agonist” is defined as a substance that has an affinity for theinactive state of a receptor and thereby preferentially stabilizes theinactive state of the receptor, or a substance, including, but notlimited to, drugs, hormones, or neurotransmitters, that producesinactivation of receptors and/or prevents or hinders activation byagonists, thereby reducing signaling from those receptors.

As used herein, the term “concurrent administration” refers to theadministration of two or more active agents sufficiently close in timeto achieve a combined therapeutic effect that is preferably greater thanthat which would be achieved by the administration of either agentalone. Such concurrent administration can be carried out simultaneously,e.g., by administering the active agents together in a commonpharmaceutically acceptable carrier in one or more doses.

The term “subject,” as used herein, refers to human or animal species.In general, methods and compositions according to the present inventioncan be used to treat not only humans, but also socially or economicallyimportant animal species such as cows, horses, sheep, pigs, goats, dogs,and cats. Unless specified, methods and compositions according to thepresent invention are not limited to treatment of humans.

The term “therapeutically effective amount,” as used herein, refers toan amount of a therapeutic agent or composition effective to treat,ameliorate, or prevent a desired disease or condition, or to exhibit adetectable therapeutic or preventative effect. The effect can bedetected by, for example, chemical markers, antigen levels, or changesin physiological indicators such as airway resistance. Therapeuticeffects also include reduction in physical symptoms, such as decreasedbronchoconstriction or decreased airway resistance, and can includesubjective improvements in well-being noted by the subjects or theircaregivers. The precise therapeutically effective amount for a subjectwill depend upon the subject's size, weight, and health, the nature andextent of the condition affecting the subject, and the therapeutics orcombination of therapeutics selected for administration, as well asvariables such as liver and kidney function that affect thepharmacokinetics of administered therapeutics. Thus, it is not useful tospecify an exact effective amount in advance. However, the effectiveamount for a given situation can be determined by routineexperimentation and is within the judgment of the clinician.

One embodiment of the invention is a method of treating a disease orcondition affected by the modulation of a beta receptor by administeringan effective quantity of an inverse agonist for the receptor whosemodulation is involved in the disease or condition. Typically, thedisease or condition is a respiratory disease or condition, including,but not limited to, asthma, chronic obstructive pulmonary disease(COPD), bronchitis, bronchiectasis, emphysema, allergic rhinitis, thepulmonary sequelae of cystic fibrosis, Churg-Strauss syndrome, andpneumonia.

In classical receptor theory, two classes of G protein-coupled receptor(GPCR) ligands were considered: agonist and antagonist. Receptors werebelieved to exist in a single quiescent state that could only inducecellular signaling upon agonist binding to produce an activated receptorstate. In this model, binding by antagonists produced no cellularsignaling but simply prevented receptors from being bound and activatedby agonists. Then, Costa and Herz demonstrated that receptors could bemanipulated into a constitutive or spontaneously active state thatproduced cellular signaling in the absence of agonist occupation. Theyalso provided evidence that certain compounds inactivate thosespontaneously active receptors (T. Costa & A. Herz, “Antagonists withNegative Intrinsic Activity at 6 Opioid Receptors Coupled to GTP-BindingProteins,” Proc. Natl. Acad. Sci. USA 86: 7321-7325 (1989)). There isfurther evidence that GPCRs exist in constitutively or spontaneouslyactive states that are inactivated to some degree by inverse agonists(R. A. de Ligt et al., “Inverse Agonism at G Protein-Coupled Receptors:(Patho)physiological Relevance and Implications for Drug Discovery,” Br.J. Pharmacol. 130:1-12 (2000); G. Milligan et al., “Inverse Agonism:Pharmacological Curiosity or Potential Therapeutic Strategy?,” TrendsPharmacol. Sci. 16:10-13 (2000)).

The basis of the strategy of this embodiment of the invention is therecognition of the existence of inverse agonists and the understandingof the effect that chronic treatment with inverse agonists has onreceptor function. What is an inverse agonist and how does it function?Receptors, such as β-adrenergic receptors that respond to adrenalin(epinephrine), typically exist in an equilibrium between two states, anactive state and an inactive state. When receptors bind to agonists,such as adrenalin for the β-adrenoceptors, they stop them from cyclingback into the inactive state, thus shifting the equilibrium between theactive and inactive states according to the Law of Mass Action. Thisoccurs because those receptors bound to agonists are removed from theequilibrium. Typically, antagonists bind to the receptors, but preventthe binding of agonists. However, molecules known as “inverse agonists”bind to the receptors in the inactive state, causing the equilibriumbetween the active and the inactive state to shift toward the inactivestate. This is not merely a matter of blocking agonist binding.

Moreover, there is a population of spontaneously active receptors invivo. These receptors provide a baseline constitutive level of activity;the activity is never entirely “off.”

As indicated above, it has been well demonstrated that chronicadministration of β-adrenergic agonists causes agonist-dependentdesensitization. Upon acute administration of β-agonists, adrenergicreceptors are internalized, thereby preventing them from beingrestimulated further for pulmonary relaxation. With chronicadministration of β-agonists, there is actually a down regulation in thetotal number of β-adrenergic receptors. The consequence may be theobserved loss of responsiveness seen in asthmatic patients onlong-acting β-agonists, and referred to as tolerance or tachyphylaxis,as described above.

The treatment methods of the present invention are based on thediscovery that a chronic administration of an inverse agonist has theeffect of up regulating the population of active β-adrenergic receptors.The observed activity may be due to the receptor's constitutive baselineactivity or the combined effect of increased level of receptorsresponding to endogenous agonists. This leads to the seeminglyparadoxical result that the administration of a drug that would appear,at first blush, to degrade a physiological function, such as by causingairway hyperresponsiveness in asthma, can, if administered chronically,enhances that physiological function by up regulating the population ofspontaneously active β-adrenergic receptors associated with thatphysiological function. This is a specific application of the principleof “paradoxical pharmacology.”

Along these lines, the use of cardioselective β inverse agonists (thosewith a preference for the β₁-adrenergic receptor subtype) has beendemonstrated to be safe in hypertensive and congestive heart failure(CHF) patients with chronic airway obstructive pulmonary disease (COPD).

Multiple studies have demonstrated that chronic administration ofcardioselective β inverse agonists does not change pulmonary function ofCHF patients with COPD or asthma. Forced expiratory volume (FEV), astandard measure of pulmonary function, was essentially unchanged inpatients treated with cardioselective β inverse agonists. These dataindicate that chronic administration of cardioselective β inverseagonists is safe in CHF patients with pulmonary airway disease. However,these drugs are not preferred for reducing or altering the symptoms ofpulmonary airway disease.

In U.S. Pat. No. 5,116,867 to Klein et al., incorporated herein by thisreference, D-propranolol or racemic mixtures composed of 85% or more ofthe D form was proposed for the treatment of asthma. The D-form ofpropranolol was 1/100 as potent as the L-form in inhibiting theβ-adrenergic receptor. In contrast, this patent specifies the use of theactive form or of racemic mixtures containing 50% or more of the activeβ-adrenergic antagonist.

In U.S. Pat. No. 6,284,800 to Broder et al., incorporated herein by thisreference, the D forms of propranolol, metoprolol, carvedilol, orbisoprolol were proposed for the treatment of asthma. Experiments wereperformed comparing the D-form versus the L-form of propranolol,demonstrating that acute administration of D-propranolol was beneficialin inhibiting antigen-induced bronchoconstriction and reducing airwayhyperresponsiveness. In contrast, acute administration of the L-formincreased specific lung resistance as expected for an activeβ-adrenergic agonist. The D form of propranolol was inactive withrespect to β-adrenergic receptors. Consequently, U.S. Pat. No. 6,284,800does not deal with inverse agonism.

PCT Patent Publication No. WO 02/29534, by Bond, had proposed compoundswith β₁ and/or β₂ antagonist activity that inhibited β-adrenergicreceptors to treat allergic and inflamatory disorders including asthmaand chronic obstructive pulmonary disease. Experiments were performed inwhich asthmatic mice were chronically treated with compoundscharacterized as β-antagonists, including alprenolol, carvedilol, andICI-118,551. Then, tracheas from the mice were excised and contractionof the tracheas in response to methacholine was monitored as a surrogatefor an asthma attack. The most effective compound was alprenolol,followed by carvedilol, then ICI-118,551.

More physiologically relevant experiments in asthmatic mice performed bythe inventor in the present application have demonstrated thatalprenolol, originally thought to be beneficial chronically, does notreduce airway hyperresponsiveness compared to untreated asthmatic mice.Even though alprenolol is a β-adrenergic antagonist, it has partialagonist activity. Carvedilol is a β₁/β₂ non-selective adrenergicantagonist with α₁-adrenergic antagonist activity. In the newexperiments reported in the present application, chronic administrationof carvedilol does reduce airway hyperresponsiveness, which would bebeneficial to asthmatics, but it also shifts the sensitivity of theresponsiveness to methacholine to lower concentrations, which could bedetrimental to asthmatics.

Moreover, in the experiments reported in PCT Patent Publication No. WO02/29534, tracheas were excised from mice, leaving behind the vastmajority of the pulmonary airways. In mice, the trachea contains almostexclusively only β₁ adrenergic receptors whereas the remainder of theairways is a mixture of β₁ and β₂ adrenergic receptors. In contrast,human airways, both the trachea and the smaller airways, contain almostexclusively β₂ receptors. Consequently, the experiments reported in PCTPatent Publication No. WO 02/29534 have little predictive value forhuman asthma. The experiments reported in the present application moreclosely reflect human physiology.

β-adrenergic antagonist drugs or “beta blockers” are treated as havingthe same activity in conventional pharmacology. Beta blockers arefurther classified based on their selectivity or lack thereof for eitherthe β₁ (termed “cardioselective”) or β₁/β₂ (“nonselective”) or β₂selective only. Additionally, beta blockers can be classified as towhether or not they have partial agonist activity or are actuallyinverse agonists. The latter definition is based on the newappreciation, recited in the present application, that many G-coupledprotein receptors, including the β-adrenergic receptors, exhibit lowlevel spontaneous activity that can be further prevented by the bindingof the inverse agonists to the receptor. This distinction was not madein PCT Patent Publication No. WO 02/29534, which referred simply to“antagonists.”

Despite this knowledge of the subclasses of beta blockers in the field,many scientists have continued to treat compounds from the differentsubclasses as one class. An example of this is the clinical testing in1998-1999 of the beta blocker bucindolol for congestive heart failure.Previously, two other beta blockers, metoprolol and carvedilol, had beenclinically tested and demonstrated significant mortality reduction inpatients with CHF. Bucindolol failed to demonstrate any benefit overplacebo, and thus clinical testing was discontinued. The inventor of thepresent application notes that both metoprolol and carvedilol areβ-inverse agonists whereas bucindolol is a neutral antagonist withpartial agonist activity. Consequently, the inventor of the presentapplication would predict that only β-adrenergic inverse agonists wouldbe effective in treatment of CHF. In the same vein, the inventor of thepresent application predicts that only β-adrenergic inverse agonistswill be effective for chronic treatment of asthma airwayhyperresponsiveness. This distinction is not made or suggested in PCTPatent Publication No. WO 02/29534.

Instead, this invention provides for the use of the active β-adrenergicreceptor binding forms of β-adrenergic inverse agonists in the treatmentof asthma, COPD, and other diseases that are marked by airwayhyperresponsiveness, including, but not limited to, emphysema,Churg-Strauss syndrome, bronchitis, and bronchiectasis. The inverseagonists can be in pure or substantially pure enantiomeric ordiastereomeric form or can be racemic mixtures. In many cases, theactive form of such compounds is the L form when there is only onechiral center. In the case of nadolol, which has three chiral centersand potentially 12 isomers, though, typically, only two are formedduring synthesis, the most active form is the RSR form of nadolol.

Especially preferred for use according to the invention are theβ-adrenergic inverse agonists: nadolol, e.g., as the hydrochloride:bupranolol, e.g., as the hydrochloride; butoxamine, e.g., as thehydrochloride; carazolol, e.g., as the hydrochloride; carvedilol; e.g.,as the hydrochloride; ICI-118,551, i.e., as the hydrochloride;levobunolol, e.g., as the hydrochloride; metoprolol, as the tartrate orsuccinate; propranolol, e.g., as the hydrochloride; sotalol, e.g., asthe hydrochloride; timolol; e.g., as the hydrochloride; and the salts,solvates, analogues, congeners, bioisosteres, hydrolysis products,metabolites, precursors, and prodrugs thereof. Particularly preferredinverse agonists are carvedilol and nadolol. A most particularlypreferred inverse agonist is nadolol. As used herein, the recitation ofan inverse agonist compound, or, where appropriate, an agonist compound,includes all pharmaceutically acceptable salts of that inverse agonistcompound or agonist compound unless excluded. Thus, the recitation ofnadolol as the hydrochloride does not exclude other pharmaceuticallyacceptable salts that have been prepared or that can be prepared.

The inverse agonists useful in methods and compositions according to theinvention typically display inverse agonism to β₂-adrenergic receptors;either as non-selective inverse agonists that display inverse agonism toboth the β₁- and β₂-adrenergic receptors or as a selective β₂-inverseagonist.

Preferably, inverse agonists useful in methods and compositionsaccording to the invention both reduce airway hyperresponsiveness and,when tested in the asthmatic mouse model, do not shift the methacholineresponse to the left (i.e., to lower methacholine concentrations).

Specifically, also expected to be within the scope of the invention areanalogues of nadolol of formula (I) wherein R₁ is hydrogen or loweralkyl, R₂ is hydrogen or lower alkyl, and m and n are 1 to 3, with theproviso that where R₁ and R₂ are both hydrogen and m is 1, n is otherthan 1. As used herein, the term “lower alkyl” is defined as a straightor branched hydrocarbyl residue of 1-6 carbon atoms.

Also specifically expected to be within the scope of the invention areanalogues of carvedilol of formula (II) wherein R₁ is hydrogen or loweralkyl, R₂ is hydrogen or lower alkyl, and R₃ is hydrogen or lower alkyl,with the proviso that all of R₁, R₂, and R₃ are not all hydrogen.

Also expected to be within the scope of the invention are analogues oftimolol of formula (III) wherein R₁ is hydrogen or lower alkyl and R₂ ishydrogen or lower alkyl, with the proviso that both R₁ and R₂ are nothydrogen.

Further expected to be within the scope of the invention are analoguesof metoprolol of formula (IV) wherein R₁ is hydrogen or lower alkyl andR₂ is hydrogen or lower alkyl, with the proviso that both R₁ and R₂ arenot hydrogen.

In the case of salts, it is well known that organic compounds, includingcompounds having activities suitable for methods according to thepresent invention, have multiple groups that can accept or donateprotons, depending upon the pH of the solution in which they arepresent. These groups include carboxyl groups, hydroxyl groups, aminogroups, sulfonic acid groups, and other groups known to be involved inacid-base reactions. The recitation of a compound or analogue includessuch salt forms as occur at physiological pH or at the pH of apharmaceutical composition unless specifically excluded.

Similarly, prodrug esters can be formed by reaction of either a carboxylor a hydroxyl group on compounds or analogues suitable for methodsaccording to the present invention with either an acid or an alcohol toform an ester. Typically, the acid or alcohol includes a lower alkylgroup such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, andtertiary butyl. These groups can be substituted with substituents suchas hydroxy, or other substituents. Such prodrugs are well known in theart and need not be described further here. The prodrug is convertedinto the active compound by hydrolysis of the ester linkage, typicallyby intracellular enzymes. Other suitable groups that can be used to formprodrug esters are well known in the art. For example prodrugs caninclude amides prepared by reaction of the parent acid compound with asuitable amine. In some cases it is desirable to prepare double estertype prodrugs such as (acyloxy) alkyl esters or((alkoxycarbonyl)oxy)alkyl esters. Suitable esters as prodrugs include,but are not necessarily limited to, methyl, ethyl, propyl, isopropyl,n-butyl, isobutyl, tert-butyl, morpholinoethyl, andN,N-diethylglycolamido. Methyl ester prodrugs may be prepared byreaction of the acid form of a compound having a suitable carboxylicacid group in a medium such as methanol with an acid or baseesterification catalyst (e.g., NaOH, H₂SO₄). Ethyl ester prodrugs areprepared in similar fashion using ethanol in place of methanol.Morpholinylethyl ester prodrugs may be prepared by reaction of thesodium salt of a suitable compound (in a medium such asdimethylformamide) with 4-(2-chloroethyl)morphine hydrochloride(available from Aldrich Chemical Co., Milwaukee, Wis. USA.

Pharmaceutically acceptable salts include acid salts such ashydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, fumarate,maleate, acetates, citrates, lactates, tartrates, sulfamates, malonate,succinate, tartrate, methanesulfonates, ethanesulfonates,benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates, quinates,formates, cinnamates, picrates, and other suitable salts. Such salts canbe derived using acids such as hydrochloric acid, sulfuric acid,phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid,tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid,benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid,and quinic acid.

Pharmaceutically acceptable salts also include salts with bases such asalkali metal salts such as sodium or potassium, as well as pyridinesalts, ammonium salts, piperazine salts, diethylamine salts,nicotinamide salts, calcium salts, magnesium salts, zinc salts, lithiumsalts, methylamino salts, triethylamino salts, dimethylamino salts, andtris(hydroxymethyl)aminomethane salts.

The subject to be treated can be a human patient or a socially oreconomically important animal, including, but not limited to, a dog, acat, a horse, a sheep, a goat, or a pig. Methods according to thepresent invention are not limited to the treatment of humans.

Typically, the method of administration of the β₂-adrenergic inverseagonist results in continuous levels of the β₂-adrenergic inverseagonist in the bloodstream of the subject. Typically, the method exertsa therapeutic effect that is an upregulation of pulmonary β₂-adrenergicreceptors. Typically, the method exerts a therapeutic effect that isincreased pulmonary airway relaxation responsiveness to β₂-adrenergicagonist drugs. This provides for combination therapy, discussed indetail below.

The β-adrenergic inverse agonist can be administered in conjunction withone or more pharmaceutical excipients. The pharmaceutical excipients caninclude, but are not necessarily limited to, calcium carbonate, calciumphosphate, various sugars or types of starch, cellulose derivatives,gelatin, vegetable oils, polyethylene glycols and physiologicallycompatible solvents. Other pharmaceutical excipients are well known inthe art. The β-adrenergic inverse agonist can be administered inconjunction with one or more pharmaceutically acceptable carriers.Exemplary pharmaceutically acceptable carriers include, but are notlimited to, any and/or all of solvents, including aqueous andnon-aqueous solvents, dispersion media, coatings, antibacterial and/orantifungal agents, isotonic and/or absorption delaying agent, and/or thelike. The use of such media and/or agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedium, carrier, or agent is incompatible with the active ingredient oringredients, its use in a composition according to the present inventionis contemplated. Supplementary active ingredients can also beincorporated into the compositions, especially as described below undercombination therapy. For administration of any of the compounds used inthe present invention, preparations should meet sterility, pyrogenicity,general safety, and purity standards as required by the FDA Office ofBiologics Standards or by other regulatory organizations regulatingdrugs.

Thus, the β-adrenergic inverse agonist can be formulated for oral,sustained-release oral, buccal, sublingual, inhalation, insufflation, orparenteral administration.

If the β-adrenergic inverse agonist is administered orally, either in aconventional or a sustained-release preparation, it is typicallyadministered in a conventional unit dosage form such as a tablet, acapsule, a pill, a troche, a wafer, a powder, or a liquid such as asolution, a suspension, a tincture, or a syrup. Oral formulationstypically include such normally employed excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, cellulose, magnesium carbonate, and other conventionalpharmaceutical excipients. In certain defined embodiments, oralpharmaceutical compositions will comprise an inert diluent and/orassimilable edible carrier, and/or they may be enclosed in hard or softshell gelatin capsules. Alternatively, they may be compressed intotablets. As another alternative, particularly for veterinary practice,they can be incorporated directly into food. For oral therapeuticadministration, they can be incorporated with excipients or used in theform of ingestible tablets, buccal tablets, dragees, pills, troches,capsules, wafers, or other conventional dosage forms.

The tablets, pills, troches, capsules, wafers, or other conventionaldosage forms can also contain the following: a binder, such as gumtragacanth, acacia, cornstarch, sorbitol, mucilage of starch,polyvinylpyrrolidone, or gelatin; excipients or fillers such asdicalcium phosphate, lactose, microcrystalline cellulose, or sugar; adisintegrating agent such as potato starch, croscarmellose sodium, orsodium starch glycolate, or alginic acid; a lubricant such as magnesiumstearate, stearic acid, talc, polyethylene glycol, or silica; asweetening agent, such as sucrose, lactose, or saccharin; a wettingagent such as sodium lauryl sulfate; or a flavoring agent, such aspeppermint, oil of wintergreen, orange flavoring, or cherry flavoring.When the dosage unit form is a capsule, it can contain, in addition tomaterials of the above types, a liquid carrier. Various other materialscan be present as coatings or to otherwise modify the physical form andproperties of the dosage unit. For instance, tablets, pills, or capsulescan be coated with shellac, sugar, or both. The pharmaceuticalcompositions of the present invention may be manufactured in a mannerthat is itself known, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levitating, emulsifying, encapsulating,entrapping or lyophilizing processes.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

In one alternative, a sustained-release formulation is used.Sustained-release formulations are well-known in the art. For example,they can include the use of polysaccharides such as xanthan gum andlocust bean gum in conjunction with carriers such as dimethylsiloxane,silicic acid, a mixture of mannans and galactans, xanthans, andmicronized seaweed, as recited in U.S. Pat. No. 6,039,980 to Baichwal,incorporated herein by this reference. Other sustained-releaseformulations incorporate a biodegradable polymer, such as the lacticacid-glycolic acid polymer recited in U.S. Pat. No. 6,740,634 to Saikawaet al., incorporated herein by this reference. Still othersustained-release formulations incorporate an expandable lattice thatincludes a polymer based on polyvinyl alcohol and polyethylene glycol,as recited in U.S. Pat. No. 4,428,926 to Keith, incorporated herein bythis reference. Still other sustained-release formulations are based onthe Eudragit™ polymers of Rohm & Haas, that include copolymers ofacrylate and methacrylates with quaternary ammonium groups as functionalgroups as well as ethylacrylate methylmethacrylate copolymers with aneutral ester group. A particularly-preferred extended releasecomposition suitable for use in methods according to the presentinvention is an extended-release composition that contains nadolol asits active ingredient.

Oral liquid preparations can be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups, tinctures, or elixirs,or can be presented as a dry product for reconstitution with water orother suitable vehicles before use. Such liquid preparations can containconventional additives such as suspending agents, for example, sorbitolsyrup, methylcellulose, glucose/sugar syrup, gelatin,hydroxymethylcellulose, carboxymethylcellulose, aluminum stearate gel,or hydrogenated edible fats; emulsifying agents, such as lecithin,sorbitan monooleate, or acacia; non-aqueous vehicles (which may includeedible oils), for example, almond oil, fractionated coconut oil, oilyesters, propylene glycol, or ethyl alcohol; or preservatives, forexample, methylparaben, propylparaben, or sorbic acid. The preparationscan also contain buffer salts, flavoring, coloring, or sweetening agents(e.g., mannitol) as appropriate.

One skilled in the art recognizes that the route of administration is animportant determinant of the rate of efficiency of absorption. Forexample, the alimentary route, e.g., oral, rectal, sublingual, orbuccal, is generally considered the safest route of administration. Thedelivery of the drugs into the circulation is slow, thus eliminatingrapid high blood levels of the drugs that could potentially have adverseacute effects. Although this is considered the safest route ofadministration, there are several disadvantages. One importantdisadvantage is that the rate of absorption varies, which is asignificant problem if a small range in blood levels separates a drug'sdesired therapeutic effect from its toxic effect, i.e., if the drug hasa relatively low therapeutic index. Also, patient compliance is notalways ensured, especially if the rectal route of administration ischosen or if oral administration is perceived by the patient asunpleasant. Furthermore, with oral administration, extensive hepaticmetabolism can occur before the drug reaches its target site. Anotherroute of administration is parenteral, which bypasses the alimentarytract. One important advantage of parenteral administration is that thetime for the drug to reach its target site is decreased, resulting in arapid response, which is essential in an emergency. Furthermore,parenteral administration allows for delivery of a more accurate dose.Parenteral administration also allows for more rapid absorption of thedrug, which can result in increased adverse effects. Unlike alimentaryadministration, parenteral administration requires a sterile formulationof the drug and aseptic techniques are essential. The most significantdisadvantage to parenteral administration is that it is not suitable forinsoluble substances. In addition to alimentary and parenteraladministration routes, topical and inhalation administrations can beuseful. Topical administration of a drug is useful for treatment oflocal conditions; however, there is usually little systemic absorption.Inhalation of a drug provides rapid access to the circulation and is thecommon route of administration for gaseous and volatile drugs, or drugsthat can be vaporized or nebulized. It is also a desired route ofadministration when the targets for the drug are present in thepulmonary system.

When compounds are formulated for parenteral administration, e.g.,formulated for injection via the intravenous, intramuscular,subcutaneous, intralesional, or intraperitoneal routes, many options arepossible. The preparation of an aqueous composition that contains aneffective amount of the β-adrenergic inverse agonist as an activeingredient will be known to those of skill in the art. Typically, suchcompositions can be prepared as injectables, either as liquid solutionsand/or suspensions. Solid forms suitable for use to prepare solutionsand/or suspensions upon the addition of a liquid prior to injection canalso be prepared. The preparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions and/or dispersions; formulations including sesame oil,peanut oil, synthetic fatty acid esters such as ethyl oleate,triglycerides, and/or aqueous propylene glycol; and/or sterile powdersfor the extemporaneous preparation of sterile injectable solutionsand/or dispersions. Aqueous injection suspensions may contain substanceswhich increase the viscosity of the suspension, such as sodiumcarboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension may also contain suitable stabilizers or agents whichincrease the solubility of the compounds to allow for the preparation ofhighly concentrated solutions. In all cases the form must be sterileand/or must be fluid to the extent that the solution will pass readilythrough a syringe and needle of suitable diameter for administration. Itmust be stable under the conditions of manufacture and storage and mustbe preserved against the contaminating action of microorganisms, such asbacteria or fungi.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and/or mixturesthereof and/or in oils. Under ordinary conditions of storage and use,these preparations contain a preservative to prevent the growth ofmicroorganisms. Suitable non-sensitizing and non-allergenicpreservatives are well known in the art.

The carrier can also be a solvent and/or dispersion medium containing,for example, water, ethanol, a polyol (for example, glycerol, propyleneglycol, and/or liquid polyethylene glycol, and/or the like), suitablemixtures thereof, and/or vegetable oils. The proper fluidity can bemaintained for example, by the use of a coating, such as lecithin, bythe maintenance of a suitable particle size in the case of a dispersion,and/or by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by the inclusion of variousantibacterial and/or antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, or thimerosal. In many cases it willbe preferable to include isotonic agents, for example, sugars or sodiumchloride. In many cases, it is preferable to prepare the solution inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiological saline buffer. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and/or gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed bysterilization. Sterilization is typically performed by filtration.Generally, dispersions are prepared by incorporating the varioussterilized active ingredients into a sterile vehicle which contains thebasic dispersion medium and/or the other required ingredients. In thecase of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying and/orfreeze-drying techniques that yield a powder of the active ingredientsplus any additional desires ingredients from a previouslysterile-filtered solution thereof. The preparation of more-concentratedor highly-concentration solutions for direct injection is alsocontemplated, where the use of dimethyl sulfoxide (DMSO) as solvent isenvisioned to result in extremely rapid penetration, delivering highconcentrations of the active agents to a small area if desired.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and/or the liquiddiluent first rendered isotonic with sufficient saline, glucose, orother tonicity agent. These particular aqueous solutions are especiallysuitable for intravenous, intramuscular, subcutaneous, orintraperitoneal administration. In this connection, sterile aqueousmedia which can be employed will be known to those of skill in the artin light of the present disclosure. For example, one dosage could bedissolved in 1 mL of isotonic NaCl solution and either added to 1000 mLof hypodermoclysis fluid or injected into the proposed site of infusion(see, e.g., “Remington's Pharmaceutical Sciences” (15^(th) ed.), pp.1035-1038, 1570-1580). Some variation in dosage will necessarily occurdepending on the condition of the subject being treated. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject. Compounds and compositionsaccording to the invention can also be formulated for parenteraladministration by bolus injection or continuous infusion and can bepresented in unit dose form, for instance as ampoules, vials, smallvolume infusions, or pre-filled syringes, or in multi-dose containerswith an added preservative.

Another route of administration of compositions according to the presentinvention is nasally, using dosage forms such as nasal solutions, nasalsprays, aerosols, or inhalants. Nasal solutions are usually aqueoussolutions designed to be administered to the nasal passages in drops orsprays. Nasal solutions are typically prepared so that they are similarin many respects to nasal secretions, so that normal ciliary action ismaintained. Thus, the aqueous nasal solutions usually are isotonicand/or slightly buffered in order to maintain a pH of from about 5.5 toabout 6.5. In addition, antimicrobial preservatives, similar to thoseused in ophthalmic preparations, and/or appropriate drug stabilizers, ifrequired, can be included in the formulation. Various commercial nasalpreparations are known and can include, for example, antibiotics orantihistamines. Spray compositions can be formulated, for example, asaqueous solutions or suspensions or as aerosols delivered frompressurized packs, with the use of a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, 1,1,1,2,3,3,3-heptafluoropropane,1,1,1,2-tetrafluoroethane, carbon dioxide, or other suitable gas.

Additional formulations that are suitable for other modes ofadministration include vaginal suppositories and/or pessaries. A rectalpessary or suppository can also be used. Suppositories are solid dosageforms of various weights or shapes, usually medicated, for insertioninto the rectum, vagina, or urethra. After insertion, suppositoriessoften, melt, and/or dissolve into the cavity fluids. In general, forsuppositories, traditional binders or carriers can include polyalkyleneglycols, cocoa butter, or triglycerides.

Other dosage forms, including but not limited to liposomal formulations,ointments, creams, lotions, powders, or creams, can alternatively beused. Ointments and creams can, for example, be formulated with anaqueous or oily base with the addition of suitable gelling agents and/orsolvents. Such bases, can thus, for example, include water and/or an oilsuch as liquid paraffin or a vegetable oil such as arachis (peanut) oilor castor oil or a solvent such as a polyethylene glycol. Thickeningagents which can be used include soft paraffin, aluminum stearate,cetostearyl alcohol, polyethylene glycols, microcrystalline wax, andbeeswax. Lotions can be formulated with an aqueous or oily base and willin general also contain one or emulsifying agents, stabilizing agents,dispersing agents, suspending agents, or thickening agents.

Powders for external application can be formed with the aid of anysuitable powder base, for example, talc, lactose, or starch.

Because of the nature of the interaction between inverse agonists andthe β-adrenergic receptors with which they interact, the therapeuticresponse develops gradually over time as the receptor density in theaffected tissues increases in response to the administration of inverseagonists. Therefore, in one particularly preferred alternative, thedosage is titrated at the start of administration with gradualincreases. In other words, the β-adrenergic inverse agonist isadministered over time in a series of graduated doses starting with thelowest dose and increasing to the highest dose. When the highest dose isreached, the β-adrenergic inverse agonist continues to be administeredat that dose (the maintenance dose). For example, with nadololadministered orally, treatment can begin with 1 mg dosages, thenprogress through 3 mg, 5 mg, 10 mg, 15 mg, and then to highermaintenance dosages such as 25 mg, 30 mg, 50 mg, 70 mg, 100 mg, orhigher as deemed necessary, depending on the particular condition to betreated, the severity, and the response of the condition to thetreatment. Analogous dosing regimens can be used with other inverseagonists, the exact starting dose typically depending on the affinity ofthe inverse agonist for the binding site of the β-adrenergic receptor.

Accordingly, another aspect of the invention is a blister pack thatincludes a range of dosages from the lowest initial dose to the highestmaintenance dose of a β-adrenergic inverse agonist such as nadolol. Ingeneral, such a blister pack comprises:

(1) a lower substrate;

(2) an intermediate dosage holder that is shaped to generate a pluralityof cavities and that is placed over the lower substrate, the cavitiesbeing shaped to hold dosage forms of a β-adrenergic inverse agonist;

(3) an upper substrate placed over the intermediate dosage holder thathas a plurality of apertures, each aperture being located to accommodatea corresponding cavity; wherein the dosage forms are of graduateddosages starting with a lowest dose and proceeding to a highest dose;and

(4) dosage forms of a β-adrenergic inverse agonist placed in thecavities.

A suitable blister pack 10 is shown in FIG. 1 and includes a lowersubstrate 12 that is typically foil, an intermediate dosage holder 14that is shaped to generate a plurality of cavities 16, 18, 20, and 22shaped to hold the pills, capsules, or other dosage forms that is placedover the lower substrate, and an upper substrate 24 placed over theintermediate dosage holder 14 that has apertures 26, 28, 30, and 32,each aperture being located to accommodate the cavities 16, 18, 20, and22. Only four cavities and apertures are shown here, but blister packs10 according to the present invention can hold a larger number of dosageforms, such as 10, 20, or 30. Typically, either the lower substrate 12,the upper substrate 24, or both have printed instructions on it toidentify the dosage of each pill, capsule, or other dosage forms, and toprovide guidance to the patient as to the sequence to be followed intaking the pills, capsules, or other dosage forms. The intermediatedosage holder 14 is typically made of a transparent plastic or othertransparent material so that the dosage forms can be viewed. The dosageforms can be of graduated doses, starting with a lowest dose andproceeding to a highest dose, which is generally the maintenance dose,as described above. Alternatively, the dosage forms can be of at leasttwo dosages: (1) a maintenance dose that is the highest in a series ofgraduated doses; and (2) at least one backup restoration dose (to beused, e.g., if a dose is missed) or a lower dose to be taken in aspecified condition. The specified condition can be, for example, theadministration of an antibiotic, such as erythromycin or neomycin, wherelower dosages are generally required or when kidney malfunctionincreases the half-life of the drug necessitating a lower dose toachieve the same serum concentration when kidney function was normal.

Various factors must be taken into account in setting suitable dosagesfor β-adrenergic inverse agonists. These factors include whether thepatient is taking other medications that can alter the pharmacokineticsof the β-adrenergic inverse agonists, either causing them to be degradedmore rapidly or more slowly. In particular, if the patient is taking theantibiotics erythromycin or neomycin, it is typically necessary todecrease the maintenance dose. Another aspect of the invention istherefore a blister pack that has backup restoration doses and lowerdoses for use when the patient is taking these antibiotics.

Toxicity and therapeutic efficacy of β-adrenergic inverse agonists canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds which exhibit large therapeutic indices arepreferred. The data obtained from these cell culture assays and animalstudies can be used in formulating a range of dosage for use in humans.The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating plasma concentration range that includes theIC₅₀ as determined in cell culture (i.e., the concentration of the testcompound which achieves a half-maximal improvement in receptor signalingwhen chronic effects are considered). Such information can be used tomore accurately determine useful doses in humans. Levels in plasma maybe measured, for example, by HPLC.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (Seee.g. Fingl et al., in The Pharmacological Basis of Therapeutics, 1975,Ch. 1 p. 1). It should be noted that the attending physician would knowhow to and when to terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministered dose in the management of the disorder of interest willvary with the severity of the condition to be treated and to the routeof administration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps the dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Typically,administration is systemic. Techniques for formulation andadministration may be found in Remington's Pharmaceutical Sciences, 18thed., Mack Publishing Co., Easton, Pa. (1990). Suitable routes mayinclude oral, rectal, transdermal, vaginal, transmucosal, or intestinaladministration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections, just to name a few. Typically, oraladministration is preferred.

For injection, the agents of the invention may be formulated in aqueoussolutions. For such transmucosal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the invention. Withproper choice of carrier and suitable manufacturing practice, thecompositions of the present invention, in particular, those formulatedas solutions, may be administered parenterally, such as by intravenousinjection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein. Inaddition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions. The pharmaceuticalcompositions of the present invention may be manufactured in a mannerthat is itself known, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levitating, emulsifying, encapsulating,entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

Typically, in methods according to the present invention, the inverseagonist is administered in a daily dose or multiple times per day,depending on the half-life of the inverse agonist. Alternatively, theinverse agonist can be administered less frequently, such as every otherday, every third day, every fourth day, every week, and the like. Oneskilled in the art of pharmacokinetics will recognize the importance ofunderstanding the bioavailability and the half-life of a drug inrelation to dosing of the particular drug. It is well known that a drugaccumulates in the body if the time interval between doses is less thanfour of its half-lives, in which case, the total body stores of the drugare increased exponentially to a plateau or steady-state concentration.The average total body store of a drug at the plateau is a function ofthe dose, the interval between doses, the bioavailability of the drug,and the rate of the elimination of the drug. Thus, one of ordinary skillin the art is capable of determining the dose and interval of the dosefor a given drug to achieve the desired effect.

Another embodiment of the present invention is methods and compositionsthat incorporate multiple-drug or combination therapy for the treatmentof pulmonary airway diseases. Patients with pulmonary airway diseasesoften are prescribed multiple drugs that work in combination to controltheir symptoms.

Although Applicant does not intend to be bound by this theory, it isbelieved that, in many circumstances, co-treatment with an inverseagonist and with an agonist is superior to treatment with the agonistalone. These results suggest that co-treatment with the inverse agonistmay increase the therapeutic efficacy of the agonist and preventdesensitization of the relevant GPCR. One rationale for this form ofcombination therapy may lie in the treatment of acute episodes such asacute asthma attacks. Even if treatment with inverse agonists decreasesthe frequency of asthma attacks, there is still a need to treat theacute attack. This can be done by co-administration of the inverseagonist and the agonist.

In one particularly desirable combination, the β-adrenergic inverseagonists are administered in combination with β₂-selective adrenergicagonists for the treatment of pulmonary airway diseases. Theβ₂-selective adrenergic agonists are typically selected from the groupconsisting of albuterol, bitolterol, clenbuterol, clorprenaline,dobutamine, fenoterol, formoterol, isoetharine, isoprenaline,levabuterol, mabuterol, metaproterenol, pirbuterol, ritodrine,salbutamol, salmeterol, and terbutaline, as well as the salts, solvates,analogues, congeners, bioisosteres, hydrolysis products, metabolites,precursors, and prodrugs thereof. A particularly preferred β-adrenergicinverse agonist according to the present invention for use in suchcombination therapy is nadolol. Particularly preferred β₂-selectiveadrenergic agonists for use in combination with β-adrenergic inverseagonists include isoproterenol, salbutamol, and salmeterol. Theprinciple of combination therapy is supported by the data that showsthat treatment with inverse agonists causes upregulation of the receptornumber. In that case, co-treatment with an agonist would be expected toincrease cellular signaling and restore normal function in thosecircumstances in which the pathological response is characterized by adeficiency in signaling. Along these lines, the inhibitory response ofinverse agonists on airway resistance would be increased in magnitude bythe co-administration of agonists. The potency of these agonists may bereduced due to the presence of the inverse agonist, but the overallmagnitude of the response would be increased. This would prevent thedesensitization often associated with chronic agonist treatment.

When combination therapy is used, the dosages of each member of thecombination can be determined according to the principles describedabove. However, in many cases, fixed dose combinations are desirable andcan be used. In the fixed dose combinations, the dosage of theβ-adrenergic inverse agonists are as described above, while thedesirable dosage of the β₂-selective adrenergic agonist can bedetermined as described above.

In another desirable combination, β-adrenergic inverse agonists areadministered together with steroids. The steroids especially preferredfor use according to the invention include, but are not necessarilylimited to, beclomethasone, budenoside, ciclesonide, flunisolide,fluticasone, methylprednisolone, prednisolone, prednisone, andtriamcinolone, as well as the salts, solvates, analogues, congeners,bioisosteres, hydrolysis products, metabolites, precursors, and prodrugsthereof.

In another desirable combination, β-adrenergic inverse agonists areadministered together with anticholinergics. The anticholinergicsespecially preferred for use according to the invention include, but arenot necessarily limited to, muscarinic receptor antagonists, especiallyquaternary ammonium muscarinic receptor antagonists such as ipratropiumbromide, tiotropium bromide, and oxitropium bromide, as well as thesalts, solvates, analogues, congeners, bioisosteres, hydrolysisproducts, metabolites, precursors, and prodrugs thereof.

In yet another desirable combination, β-adrenergic inverse agonists areadministered together with a xanthine compound. Xanthine compoundsespecially preferred for use according to the invention include, but arenot necessarily limited to, theophylline, extended-release theophylline,aminophylline, theobromine, enprofylline, diprophylline, isbufylline,choline theophyllinate, albifylline, arofylline, bamifylline andcaffeine, as well as the salts, solvates, analogues, congeners,bioisosteres, hydrolysis products, metabolites, precursors, and prodrugsthereof.

In yet another desirable combination, β-adrenergic inverse agonists areadministered together with an anti-IgE antibody. Typically, the anti-IgEantibody is a monoclonal antibody or a genetically engineered antibodythat is derived from a monoclonal antibody. Preferably, the anti-IgEantibody is humanized. A particularly preferred humanized anti-IgEantibody is an IgG1 κ monoclonal antibody that specifically binds tohuman IgE and is marketed under the name of omalizumab.

In still another desirable combination, β-adrenergic inverse agonistsare administered together with a leukotriene modifier. The leukotrienemodifiers especially preferred for use according to the presentinvention include, but are not necessarily limited to, ibudilast,montelukast, pranlukast, and zafirlukast, as well as the salts,solvates, analogues, congeners, bioisosteres, hydrolysis products,metabolites, precursors, and prodrugs thereof.

In still another desirable combination, β-adrenergic inverse agonistsare administered together with a phosphodiesterase IV inhibitor. Thephosphodiesterase IV inhibitors especially preferred according to thepresent invention include, but are not necessarily limited to,roflumilast and cilomilast, as well as the salts, solvates, analogues,congeners, bioisosteres, hydrolysis products, metabolites, precursors,and prodrugs thereof. Phosphodiesterase IV is the predominant isoform inthe lung and inhibitors of this enzyme are being considered for thetreatment of asthma and COPD.

The route of administration of the β-adrenergic inverse agonist and ofthe additional therapeutic agent can be chosen by one of ordinary skillin the art to optimize therapeutic efficiency, as described above.However, in one preferred alternative, both the β-adrenergic inverseagonist and the additional therapeutic agent are administered byinhalation. In another preferred alternative, the β-adrenergic inverseagonist is administered orally, while the additional therapeutic agentis administered by inhalation. The administration of the additionaltherapeutic agent by inhalation is typically preferred because ofpossible toxicity of some of these additional therapeutic agents.However, other routes are possible.

Aerosol therapy allows an almost ideal benefit to risk ratio to beachieved because very small doses of inhaled medication provide optimaltherapy with minimal adverse effects. A variety of additionaltherapeutic agents suitable for use in methods according to the presentinvention are available in aerosol formulation, including β₂-adrenergicagonists, steroids, and anticholinergics. However, the therapeuticefficiency of drugs administered by aerosolization depends not only onthe pharmacological properties of the drugs themselves, but also on thecharacteristics of the delivery device. The characteristics of thedelivery device influence the amount of drug deposited in the lungs andthe pattern of drug distribution in the airways.

Aerosols are airborne suspensions of fine particles. The particles maybe solids or liquids. Aerosol particles are heterodisperse (i.e. theparticles are of a range of sizes) and aerosol particle sizedistribution is best described by a log normal distribution. Particlestend to settle (sediment), adhere to each other (coagulate), and adhereto structures such as tubing and mucosa (deposit). The particlesdelivered by aerosol can be conveniently characterized on the basis oftheir aerodynamic behavior. One parameter is the mass median aerodynamicdiameter (MMAD). By definition, a particle distribution with an MMAD of1 μM has the same average rate of settling as a droplet of unit densityand 1 μM diameter.

The size of an aerosol particle, as well as variables affecting therespiratory system, influence the deposition of inhaled aerosols in theairways. On one hand, particles larger than 10 μM in diameter areunlikely to deposit in the lungs. However, particles smaller than 0.5 μMare likely to reach the alveoli or may be exhaled. Therefore, particlesthat have a diameter of between 1 μM and 5 μM are most efficientlydeposited in the lower respiratory tract. Particles of these sizes aremost efficient for the delivery of therapeutic agents for asthma.

The percentage of the aerosol mass contained within respirable droplets(i.e., droplets with a diameter smaller than 5 μM), depends on theinhalation device being used. Slow, steady inhalation increases thenumber of particles that penetrate the peripheral parts of the lungs. Asthe inhaled volume is increased, the aerosol can penetrate moreperipherally into the bronchial tree. A period of breath-holding, oncompletion of inhalation, enables those particles that have penetratedto the lung periphery to settle into the airways via gravity. Increasedinspiratory flow rates, typically observed in patients with acuteasthma, result in increased losses of inhaled drug. This occurs becauseaerosol particles impact in the upper airway and at the bifurcations ofthe first few bronchial divisions. Other factors associated withpulmonary airway disease may also alter aerosol deposition. Airwayobstruction and changes in the pulmonary parenchyma are often associatedwith pulmonary deposition in the peripheral airways in patients withasthma.

In aerosol administration, the nose efficiently traps particles beforetheir deposition in the lung; therefore, mouth breathing of theaerosolized particles is preferred. The aerosolized particles are lostfrom many sites. Generally, the amount of the nebulized dose reachingthe small airways is ≦15%. In many cases, approximately 90% of theinhaled dose is swallowed and then absorbed from the gastrointestinaltract. The small fraction of the dose that reaches the airways is alsoabsorbed into the blood stream. The swallowed fraction of the dose is,therefore, absorbed and metabolized in the same way as an oralformulation, while the fraction of the dose that reaches the airways isabsorbed into the blood stream and metabolized in the same way as anintravenous dose.

When drugs are administered topically (via aerosol delivery to thelungs), the desired therapeutic effects depend on local tissueconcentrations, which may not be directly related to plasma drugconcentrations. If a sufficiently large dosage of any drug is given,systemic activity can easily be demonstrated with any inhaledβ₂-agonists or corticosteroid. This has several implications. First, forthe selection of a drug to be inhaled, topical drugs must combine a highintrinsic activity within the target organ and rapid inactivation of thesystemically absorbed drug. Secondly, fewer systemic adverse effectsshould be expected with drugs that have a low oral bloavailability(whether due to poor gastrointestinal absorption or high first-passhepatic metabolism). Because most inhaled drugs are administered at alow dosage and have a low oral bioavailability, plasma concentrations ofthese drugs are much lower than after oral administration.

Furthermore, factors influencing pulmonary absorption should beconsidered. It was recently demonstrated that terbutaline was absorbedthrough the lung more rapidly in healthy smokers than in healthynonsmokers. This may affect the onset of action of the drug. It has alsobeen found that the bioavailability of inhaled salbutamol in 10 patientswith cystic fibrosis was greater than that in healthy adults. Oneproposed mechanism for this difference in bioavailability is that thechronically diseased tracheobronchial tree in patients with cysticfibrosis results in higher permeability of salbutamol in this tissue.However, data are limited in this area, and further investigation isrequired to substantiate these claims.

Finally, the absolute pulmonary bioavailability of inhaled drugs isdifficult to assess because blood concentrations are low, and pulmonaryand oral absorption should be discriminated for pulmonarybioavailability to be determined as accurately as possible. Charcoal canbe used to adsorb the swallowed fraction of inhaled terbutaline todiscriminate the pulmonary absorption of the drug. Recently, it wasshown that a urine collection during the 30 minutes after inhalation ofsalbutamol represents the amount of drug delivered to the lungs. Thistechnique may be applicable for the determination of bioavailability ofother inhaled drugs. Other techniques for the determination ofbioavailability of inhaled drugs are also known in the art; theseinclude pharmacodynamic methods using FEV₁ measurements, lung depositionstudies using radiolabeled formulations, or pharmacokinetic studiesusing predominantly urinary excretion measurements.

Therapeutic aerosols are commonly produced by atomization of liquidswithin jet nebulizers or by vibration of a standing pool of liquid(ultrasonic nebulisation). Preformed aerosols may also be administered.Examples of the latter include MDIs and dry powder devices. Whateverdelivery device is used, patients should be taught to use it correctly.

All jet nebulizers work via a similar operating principle, representedby the familiar perfume atomizer. A liquid is placed at the bottom of aclosed container, and the aerosol is generated by a jet of air fromeither a compressor or a compressed gas cylinder passing through thedevice. Ultrasonic nebulizers produce an aerosol by vibrating liquidlying above a transducer at frequencies of about 1 mHz. This produces acloud of particles that is carried out of the device to the patient by astream of air. Aerosols varying in quantity, size and distribution ofpanicles can be produced by nebulizers, depending upon the design of thenebulizers and how it is operated. It should be noted that not allnebulizers have the required specifications (MMAD, flow, output) toprovide optimum efficacy. A recent study compared the lung depositionfrom 4 nebulizers in healthy volunteers and showed that median lungaerosol deposition, expressed as percentages of the doses initiallyloaded into the nebulizers, ranged from 2 to 19%. Nebulized aerosols areparticularly useful for children under 5 years of age and in thetreatment of severe asthma where respiratory insufficiency may impairinhalation from an MDI or dry powder inhaler. To minimize adverseeffects, pH and osmolarity of the nebulized solution should becontrolled.

Metered dose inhalers (MDIs), because of their convenience andeffectiveness, are probably the most widely used therapeutic aerosolused for inhaled drug delivery to outpatients. Most MDIs in current usecontain suspensions of drug in propellant. There are 2 major componentsof an MDI: (i) the canister, a closed plastic or metal cylinder thatcontains propellant, active medication, and the metering chamber; and(ii) the actuator, a molded plastic container that holds the canisterand directs the released aerosol towards the patient's airway.

Propellant mixtures are selected to achieve the vapor pressure and spraycharacteristics desired for optimal drug delivery. Chlorofluorocarbonswere previously used, but non-chlorinated propellants are now employedbecause of environmental concerns. Finely divided particles of drug,usually less than 1 μM, are suspended in the pressurized (liquefied)propellant. To prevent the drug from coagulating, a surface active agentsuch as sorbitan oleate, lecithin or oleic acid is typically added;other surface active agents are known in the art. Metering chambersordinarily contain 25 to 100 μL. The contents of the metering chamberare released when the canister is depressed into the actuator. Almostinstantaneously, the propellants begin to evaporate, producingdisintegration of the discharged liquid into particles that arepropelled forward with great momentum. For optimal pulmonary drugdeposition, the medication should be released at the beginning of a slowinspiration that lasts about 5 seconds and is followed by 10 seconds ofbreath-holding. Several inhalation aids have been designed to improvethe effectiveness of a MDI. These are most useful in patients who havepoor hand-to-breath coordination. A short tube (e.g. cones or spheres)may direct the aerosol straight into the mouth or collapsible bags mayact as an aerosol reservoir holding particles in suspension for 3 to 5seconds, during which time the patient can inhale the drug. However,when any of these devices is used, aerosol velocity upon entering theoropharynx is decreased and drug availability to the lungs anddeposition in the oropharynx is decreased.

Dry powder inhalers have been devised to deliver agents to patients whohave difficulty using an MDI (e.g. children and elderly patients). Ingeneral, the appropriate dosage is placed in a capsule along with a flowaid or filler such as large lactose or glucose panicles. Inside thedevice, the capsule is initially either pierced by needles (e.g.Spinhaler®) or sheared in half (e.g. Rotohaler®). During inhalation thecapsule rotates or a propeller is turned, creating conditions that causethe contents of the capsule to enter the inspired air and be broken upto small particles suitable for delivery to the airways. The energyrequired to disperse the powder is derived from the patient'sinspiratory effort. Recently, more convenient multidose dry powderinhalers have been introduced (e.g. Diskhaler®, Turbuhaler®)). Potentialproblems associated with dry powder inhalers include esophagealirritation and, consequently, cough due to the direct effect of powderin airways. Furthermore, the walls of the capsule may be coated withdrug as a result of either failure of the capsule to release the drug orfailure of the aggregated powder to break up. This may cause virtuallyall of the drug to be deposited in the mouth. These powder devices donot contain chlorofluorocarbons and may provide an alternative to MDIs.

The clinical use of aerosols for asthma treatment has been proposed forseveral compounds proposed herein as additional therapeutic agents,including β₂-agonists and corticosteroids.

For β₂-agonists, limited pharmacokinetic data are available in humansmostly because the low dosages of inhaled drugs required for therapeuticactivity produce drug concentrations in body fluids that are below assaylimits. Little is known about pulmonary bioavailability of those drugs.It is generally argued that an average of 10% of an inhaled dose reachesthe lung when given by a MDI. The mean pulmonary bioavailability ofterbutaline from an MDI was reported to be 9.1%. When the oral component(swallowed fraction of the dose) was added, the value rose to 16.5%,i.e. an increase of 6.7%. The drugs salmeterol and formoterol havedifferent mechanisms of action underlying their prolonged duration ofbronchodilatory effect (12 to 18 hours). Salmeterol appears uniquebecause it has a long side-chain that anchors the β₂-agonist molecule tothe receptor. Formoterol appears to be an extremely potent classicalβ₂-agonist. The elimination half-life of formoterol after inhalation wascalculated to be between 1.7 and 2.3 hours on the basis of urinaryexcretion data. A glucuronic acid conjugate was identified. However, itis possible that formoterol has a prolonged elimination half-life thatis yet to be detected in humans. Salmeterol is formulated as thexinafoate (hydroxynaphthoic acid) salt. Little is known about thepharmacokinetic properties of this drug Salmeterol is extensivelymetabolized by hydroxylation, with the majority of a dose beingeliminated predominantly in the feces within 72 hours. Thehydroxynaphthoic acid part of the molecule accumulates in plasma duringrepeated administration as a consequence of its long eliminationhalf-life (12 to 15 days).

For anticholinergic agents, the parent compound of this class isatropine. Synthetic agonists of the muscarinic receptors ofacetylcholine are quaternary ammonium compounds and, therefore, crossmembrane barriers with difficulty. Because systemic absorption ofatropine after inhalation of the drug is nearly complete, this route ofadministration can produce significant systemic toxicity (Harrison etal. 1986). Ipratropium bromide is the only well studied representativeof this class. Absorption through the gastrointestinal tract is slow, aspeak plasma concentrations have been recorded 3 hours after oral intakeof the drug. The absolute bioavailability after oral intake is only 30%.Elimination of metabolized drug occurs in the urine and bile. Whateverthe route of administration, the mean elimination half-life is about 3.5hours. Plasma concentrations observed with inhaled ipratropium were athousand times lower than those observed with an equipotentbronchodilatory dose administered orally. This explains why systemicanticholinergic effects do not occur following inhalation of therapeuticdoses of ipratropium bromide. These properties are probably shared byother quaternary ammonium anticholinergic agents such as oxitropiumbromide, an alternative as described above.

Corticosteroids are frequently administered by inhalation, which canprevent some of the adverse effects usually associated with systemiccorticosteroid therapy. To produce a compound with marked topicalactivity, some of the hydroxyl groups in the hydrocortisone moleculewere substituted with acetonide or ester groups. Topically activecorticosteroid drugs used for the treatment of patients with asthmainclude beclomethasone, betamethasone valerate, budesonide,triamcinolone, fluticasone and flunisolide. Of these, beclomethasone andbudesonide are the most extensively used. The results of numerousclinical studies have shown that there is little difference between theefficacy of beclomethasone and budesonide. Oropharynx deposition isreduced by using a spacing device, and the development of candidiasiscan be prevented by mouth rinsing. Plasma clearance of budesonide wascalculated to be 84±27 L/h, which is about 10-fold higher than theaverage clearance of prednisolone. As a consequence of this highclearance, the elimination half-life of budesonide is short (2.8±1.1hours). The systemic availability of the swallowed fraction is10.7±4.3%, indicating that there is extensive first-pass metabolism.Stereoselective metabolism was demonstrated and plasma clearance of the2 enantiomers, when calculated on a per kilogram of bodyweight basis,were about 50% higher in 6 children with asthma than in 11 healthyadults. Therefore, administration of budesonide by inhalation shouldreduce the risk of systemic adverse effects compared with administrationof the drug orally. Lung esterases are known to hydrolyzebeclomethasone. The absorbed beclomethasone and esterase-hydrolysisproducts (beclomethasone 17-propionate and beclomethasone) are rapidlyconverted to less active metabolites during passage through the liver.First-pass hepatic metabolism of the systemically absorbed fluticasoneis almost complete, and therefore the inhaled drug has a favorablepharmacokinetic profile. Few data have been published regarding thepharmacokinetic properties of flunisolide, triamcinolone andbetamethasone valerate.

To ensure maximal effects from inhaled drugs, both the pharmacologicalcharacteristics of the drugs and the device used to aerosolize the drugsshould be considered. With respect to β₂-agonists, differentformulations, with different pulmonary disposition techniques, areavailable, such as for MDI administration, for administration with a drypowder inhaler, or a solution for nebulisation. A unit dose from a drypowder inhaler is twice that release from an MDI, but they haveequivalent bronchodilatory effects. The characteristics of the devicesvary. For a metered-dose inhaler, typically 12-40% of the dose isdeposited in the lung, but up to 80% in the oropharynx. When an MDI isused with a spacer, typically about 20% of the dose is deposited in thelung, but only up to 5% in the oropharynx; thus, the use of a spacer canreduce the proportion of the drug that is deposited in the oropharynx.For a dry powder inhaler, typically 11-16% of the dose is deposited inthe lung and 31-72% in the oropharynx. For a nebulizer, typically 7-32%of the dose is deposited in the lung and 1-9% is deposited in theoropharynx. One of ordinary skill in the art can ensure that the properinhalation therapy device is used and can prepare suitable instructions.Considerations for the use of inhalation therapy are described in A.-M.Tabaret & B. Schmit, “Pharmacokinetic Optimisation of Asthma Treatment,”Clin. Pharmacokinet. 26: 396418 (1994), incorporated herein by thisreference.

For all of these combinations, the invention further encompasses blisterpacks that contain either a fixed-dose combination of the β-adrenergicinverse agonist and the additional therapeutic agent, such as theβ₂-selective adrenergic agonist, the steroid, the anticholinergic agent,the xanthine compound, the anti-IgE antibody, the leukotriene modifier,or the phosphodiesterase-4 inhibitor, or, in separate pills, capsules,or other dosage forms, the β-adrenergic inverse agonist and theadditional therapeutic agent. The use of these blister packs isappropriate when oral administration of the inverse agonist andadditional therapeutic agent is desired. The blister packs follow thegeneral design described above and in FIG. 1, and include appropriateinstructions to the patient.

In general, when a fixed-dose combination is used, the blister packcomprises:

(1) a lower substrate;

(2) an intermediate dosage holder that is shaped to generate a pluralityof cavities and that is placed over the lower substrate, the cavitiesbeing shaped to hold dosage forms of the pharmaceutical compositiondescribed above containing a β-adrenergic inverse agonist and anadditional therapeutic agent;

(3) an upper substrate placed over the intermediate dosage holder thathas a plurality of apertures, each aperture being located to accommodatea corresponding cavity; and

(4) dosage forms of the pharmaceutical composition placed in thecavities.

When the β-adrenergic inverse agonist and the additional therapeuticagent are to be administered in separate dosage forms, the blister pack,in general, comprises:

(1) a lower substrate;

(2) an intermediate dosage holder that is shaped to generate a pluralityof cavities and that is placed over the lower substrate, the cavitiesbeing shaped to hold dosage forms of: (a) a first pharmaceuticalcomposition that comprises: (i) a therapeutically effective amount of aβ-adrenergic inverse agonist; and (ii) a first pharmaceuticallyacceptable carrier; and (b) a second pharmaceutical composition thatcomprises: (i) a therapeutically effective amount of a secondtherapeutic agent effective to treat a pulmonary airway disease, thesecond therapeutic agent being selected from the group consisting of aβ₂-selective adrenergic agonist, a steroid, an anticholinergic drug, axanthine compound, an anti-IgE antibody, a leukotriene modifier, and aphosphodiesterase IV inhibitor; and (ii) a second pharmaceuticallyacceptable carrier;

(3) an upper substrate placed over the intermediate dosage holder thathas a plurality of apertures, each aperture being located to accommodatea corresponding cavity; and

(4) dosage forms of the first and second pharmaceutical compositionsplaced in the cavities.

The dosage forms of the first and second pharmaceutical compositions areas described above. Typically, in this arrangement, the dosage forms ofthe first pharmaceutical composition include dosages starting at a lowdose and including a range of dosages up to the highest, maintenance,dose. Other dosage form arrangements are possible.

Other arrangements are possible for the blister packs.

The invention is illustrated by the following Examples. These Examplesare included for illustrative purposes only, and are not intended tolimit the invention.

EXAMPLES Example 1 Airway Resistance Reduction by Chronic Administrationof β-Adrenergic Inverse Agonists

Methods

Balb/cJ mice aged 6 weeks (Jackson Animal Laboratory, Bar Harbor, Me.)were housed under specific pathogen-free conditions and fed a chickenovalbumin-free diet. The Animal Research Ethics Boards of both theUniversity of Houston and the Baylor College of Medicine approved allexperiments reported here. The effects of administration of thenon-selective β-adrenergic inverse agonists carvedilol (GlaxoSmithKline,King of Prussia, Pa.) and nadolol (Sigma Chemical, St. Louis, Mo.) andof salbutamol (Sigma Chemical, St. Louis, Mo.), a β₂-adrenergic partialagonist, were examined in a murine model that exhibited cardinalfeatures of human asthma, such as pulmonary eosinophilic inflammation,airway hyperresponsiveness, and heterogeneous airway narrowing. Theresults obtained in drug-treated animals were compared with thoseobtained in vehicle-treated counterparts (controls) in experimentsperformed in close temporal relationship. The outcome measures of thestudy of Example 1 included statistically-significant differencesbetween drug-treated mice and non-treated animals in terms of baselineairway resistance, degree of airway responsiveness to cholinergicstimulation, and bronchoalveolar lavage (BALF) cellularity. Mice weresensitized with subcutaneous injection of 25 μg of ovalbumin adsorbed toaluminum hydroxide on protocol days 2, 9, and 16. Subsequently, micewere given 50 μL of saline solution containing 25 μg of ovalbuminintranasally, on a daily basis, from protocol days 23 through 27. Agroup of ovalbumin-sensitized saline-challenged mice serves as controlsfor systemic sensitization and respiratory challenges with ovalbumin.Prior to intranasal administrations, mice were sedated with halothanevapor. For the study of Example 1, ovalbumin-sensitized andovalbumin-challenged mice, and ovalbumin-sensitized andsaline-challenged mice will be referred to as asthmatic mice and controlmice, respectively. The drugs used were salbutamol (a β₁/β₂-adrenergicagonist), alprenolol (a β₁/β₂-adrenergic antagonist with partial agonistactivity), and nadolol and carvedilol (both non-selective β₁/β₂adrenergic inverse agonists).

To examine the effects of duration of β-adrenergic ligand therapy on thephenotype of the murine model of asthma, the experimental drugs wereadministered either acutely or chronically to different groups ofasthmatic mice.

Asthmatic mice on acute therapy were given a single intravenous bolusinfusion of either β-adrenergic drug or normal saline on protocol day28, 15 minutes before airway responsiveness to methacholine wasdetermined. The doses of carvedilol, nadolol, alprenolol, and salbutamoladministered to the mice were 24 mg/kg, 72 mg/kg, 72 mg/kg, and 0.15mg/kg, respectively. Asthmatic mice on chronic therapy were treated withthe β-adrenergic drug during protocol days 1 to 28. Those onβ-antagonists had free access to chow treated with carvedilol, nadolol,or alprenolol at concentrations of 2400 ppm, 250 ppm, or 7200 ppm,respectively. These concentrations were chosen based on those producingtherapeutic effects in mice in previously published studies. Thenon-asthmatic mice were fed normal chow. Salbutamol was delivered for 28days at a dose of 0.5 mg/kg/day using an osmotic minipump (Alzet®,#2004, Durect Corporation, Cupertino, Calif.).

On protocol day 28, mice were anesthetized, tracheotomized, andconnected to a computer-controlled small animal ventilator (Flexivent,Scientific Respiratory Equipment, Inc., Montreal, Canada). Airwayresistance (R_(aw)) was measured using the forced oscillation technique.The cellular composition of bronchoalveolar lavage fluid (BALF) was alsodetermined. In non-treated asthmatic mice, the degree of airwayresponsiveness and the number of eosinophils recovered in BALF weresignificantly higher compared to the ovalbumin-sensitizedsaline-challenged (control) mice. However, it was observed that thedegree of airway responsiveness and the number of eosinophils recoveredin BALF were lower in non-treated asthmatic mice studied in closetemporal relationship with mice receiving acute β-adrenergic antagonisttreatments that in those obtained in non-treated asthmatic mice studiedconcomitantly with mice on chronic β-adrenergic antagonist therapy.

To induce airway constriction, a solution containing 150 μg/mL ofacetyl-α-methylcholine chloride (methacholine) (Sigma Chemical, St.Louis, Mo.) was infused intravenously at constant rates using a syringeinfusion pump (Raze Scientific Instruments, Stanford, Conn.). Themethacholine infusion was started at 0.008 mL/min, and its rate wasdoubled stepwise up to a maximum of 0.136 mL/min. Each methacholine dosewas administered for 3 to 5 minutes, during which data were sampled at1-minute intervals and then averaged.

Data Analysis

The complex input impedance of the respiratory system was computed andthe value of the real part of respiratory system impedance at 19.75 Hzwas taken to reflect the magnitude of airway resistance (R_(aw)). Toexamine the degree of airway responsiveness of each animal, the valuesfor R_(aw) as a function of methacholine doses were plotted. The largestvalue for R_(aw) achieved in response to methacholine stimulation wasreferred to as R_(awpeak). For mice that achieved a plateau in themethacholine dose-R_(aw) response curve, the ED₅₀ was calculated bylinear interpolation using the GraphPad Prism4 (GraphPad Software,Inc.). Results obtained for β-adrenergic drug treated and non-treatedmice were performed using the analysis of variance for multiple groupsof a Student's t-test for comparing two groups. The Bonferroni test wasused to examine the statistical differences between experimental groups.The effects of acute drug treatments on baseline respiratory systemmechanics were assessed using a two-tailed paired t-test. A value ofP<0.05 was considered significant.

FIG. 2

FIGS. 2A and 2B show that methacholine provocation significantlyenhances airway resistance (R_(aw)) in asthmatic mice in contrast to aminimal response upon saline provocation of asthmatic mice. Thisdemonstrates that the mouse model in this study exhibits airwayhyperresponsiveness, a key feature of airway dysfunction in humanasthma.

In FIG. 2C, the administration of a single intravenous bolus ofsalbutamol to asthmatic mice reduced the level of airway responsivenessto methacholine provocation and the level of airway resistance asexpected, thus demonstrating an acute effect of this agent. However, inFIG. 2D, when salbutamol was delivered for 28 days to the mice, noprotection was observed. This lack of reduction of airwayhyperresponsiveness upon chronic administration of a β-adrenergicagonist has been observed in humans when tolerance to these drugsdevelop.

In FIG. 2E, when asthmatic mice were given a single intravenous bolus ofalprenolol, a β-adrenergic antagonist with partial agonist activity,their airway responsiveness was diminished, as indicated by significantdecreases in both the values for R_(aw) at methacholine doses ≧408μg/kg/min (P<0.05) compared with those obtained in non-treatedcounterparts. The reduction in airway responsiveness upon acuteadministration of alprenolol is similar to that observed for salbutamol,consistent with the partial agonist activity that alprenolol possesses.In FIG. 2F, when asthmatic mice were exposed to alprenolol for 28 days,their average methacholine dose-response relationship was similar tothat obtained in nontreated mice, demonstrating that this drug providesno benefit upon chronic administration, as is the case with salbutamol.This is again directly analogous to the tolerance seen in human patientsafter long-term administration of such drugs.

In FIG. 2G, a single intravenous bolus of carvedilol enhanced the airwayresponsiveness in the asthmatic mice. This is consistent with previousobservations in humans that acute delivery of β-adrenergic antagoniststo asthmatics can result in severe airway constriction. In contrast, inFIG. 2H, chronic administration of carvedilol reduced the responsivenessof asthmatic mice to methacholine provocation.

In FIG. 2I, a single intravenous bolus of nadolol also enhanced theairway responsiveness of asthmatic mice similar to that observed forcarvedilol. Chronic delivery of nadolol, as shown in FIG. 2J, alsoproduced a decrease in airway responsiveness, which was more pronouncedthan that caused by carvedilol treatment. Indeed, the averagemethacholine dose-R_(aw) response relationship obtained in asthmaticmice on chronic nadolol treatment was similar to that obtained in miceon acute salbutamol treatment.

FIG. 3

FIG. 3 shows the effects of administration of β-adrenergic receptorligands on the peak airway responsiveness to cholinergic stimulation inasthmatic mice. Peak R_(aw) was determined for each mouse by examiningthe individual methacholine dose-response curves and choosing thehighest R_(aw) value produced by any of the methacholine doses (mostoften the next to last dose, 408 μg kg⁻¹ min⁻¹). Shown are the mean peakR_(aw)±SEM after treatments with the β-adrenergic receptor agonistsalbutamol (A), after acute treatments with various agents (B)(ALP=alprenolol; CAR=carvedilol; NAD; nadolol); and after chronictreatments with the same agents used in (B), all in comparison tonontreated asthmatic mice (NTX) (black bars, n=7-25) and control mice(Ctrl, white bars, n=6-21). Values are mean±SEM for the peak R_(aw)values to methacholine of n=8-19 mice. Note the change in scale of they-axis for (B). *, P<0.05 compared to NTX; #, P<0.05 compared to Ctrl(ANOVA).

Example 2 Chronic Inverse Agonist Treatment Increases β-AdrenergicReceptor Numbers as Measured by Radioligand Binding

β₂-adrenergic receptor numbers were measured in asthmatic mice asfollows. Asthmatic mice (ovalbumin-challenged) were treated as follows:Ctrl, no drug treatment with methacholine challenge; salbutamol, ashort-acting β₂ agonist; carvedilol, a β₁, β₂ non-selective inverseagonist with α₁-adrenergic antagonist activity; nadolol, a highlyspecific, hydrophilic β₁, β₂ non-selective inverse agonist; andalprenolol, a β-adrenergic antagonist. Drug treatments were either asingle treatment 15 minutes prior to methacholine challenge or ongoingfor 28 days (salbutamol was delivered continuously via a subcutaneousosmotic minipump and alprenolol, carvedilol, and nadolol were in animalchow). Mice were sacrificed and lung membranes were isolated as follows.Frozen lung tissue was homogenized in an ice-cold buffer containing 0.32M sucrose and 25 mM Tris (pH 7.4) using a polytron (Pro 200, ProScientific, Inc.). The homogenate was centrifuged at 1000×g for 10 minat 4° C. The resulting supernatant was centrifuged at 40,000×g for 20min at 4° C. The pellet was suspended in an ice-cold 25 mM Tris-HClbuffer (pH 7.4) and centrifuged at 40,000×g for 20 min at 4° C. Thefinal pellet was suspended in 200 μL of 25 mM Tris-HCl (pH 7.4);membrane protein concentration was determined by BCA protein assay kit.Radioligand receptor binding incubation mixtures contained membranes(˜10 μg of protein), (−)3-[¹²⁵I]-cyanopindolol (ICYP) in 25 mM Tris-HCl,pH 7.4, in increasing concentrations (57500 μM) and binding buffer in afinal volume of 250 μL. Propranolol was used to determine nonspecificbinding. The incubation was done at 37° C. for 2 h and terminated byrapid vacuum filtration through glass fiber filters. The filters werewashed three times with 250 μL of cold wash buffer (25 mM Tris-HCl, pH7.4) and the radioactivity determined in a counter. All experiments wereperformed in triplicate and values are mean±SEM of n=3-5 animals in eachgroup. Receptor densities are expressed as femtomoles of sites permilligram of protein. B_(max) is determined by nonlinear regression ofthe saturation binding curves. Apparent K_(D) values (in parentheses)are expressed as pM. Please note the 15 min and 28 day tome points referto duration of drug treatment. All mice were killed at the same age andthus for vehicle treated groups (Ctrl and NTX) the groups were identicaland the results pooled. #P<0.05 compared to Ctrl; *P<0.05 compared toNTX (Student's t-test).

Radioligand binding revealed that β₂-adrenergic receptor levels appearto be somewhat lower in methacholine-challenged but otherwise untreatedasthmatic mice as compared with untreated, unchallenged mice, as shownin Table 1. Chronic alprenolol treatment led to a slight decrease of thelevel of the β₂-adrenergic receptor. The same was true of chronicsalbutamol treatment. Most significantly, the carvedilol-treated micedemonstrated an over 10-fold increase of the level of β₂-adrenergicreceptors over the non-treated mice, demonstrating the efficacy of thisβ-adrenergic inverse agonist in increasing receptor levels upon chronicadministration. Similarly, the nadolol-treated mice demonstrated anearly eightfold increase of the level of receptors over the untreatedmethacholine-challenged asthmatic mice.

TABLE 1 Determination of β-Adrenergic Receptor Density by RadioligandBinding 15 Minutes 28 Days Treatment B_(max) K_(D) β_(max) K_(D) Ctrl286.8 ± 88.02 (107.9 ± 43.67) 286.8 ± 88.02 (107.9 ± 43.67) NTX 109.2 ±9.72# (193.6 ± 20.66) 109.2 ± 9.72# (193.6 ± 20.66) Salbutamol  256.5 ±29.24* (228.8 ± 33.07)  97.0 ± 23.02 (225.4 ± 41.79) Alprenolol  299.5 ±12.19* (453.6 ± 86.33) 179.2 ± 53.05 (290.9 ± 55.07) Carvedilol  86.3 ±19.42  (565.2 ± 192.8)*  904.1 ± 43.46* (1444.0 ± 202.0)  Nadolol 181.9± 48.28  (695.1 ± 286.3)*  785.5 ± 154.8* (1591.6 ± 335.0)*

Example 3 Chronic Inverse Agonist Treatment Increases β-AdrenergicReceptor Numbers as Monitored by Immunohistochemistry

For immunohistochemistry analysis of β₂-adrenergic receptor levels,non-drug-treated control mice and mice treated chronically with theβ₂-adrenergic inverse agonist nadolol were used. The mice weresacrificed and the lungs excised. Then the lungs were fixed in 4%paraformaldehyde (45 min, 0° C.). After fixation, lungs were washed inPBS (60 min) and placed in increasing concentrations of sucrose (10%sucrose/5% glycine in PBS for 30 min; 20% sucrose/10% glycine in PBS for30 min; 30% sucrose/15% glycine in PBS for 12 h at 4° C.). Lungs wereembedded in OCT and 12-μm sections cut with a Tissue-Tek II cryostat.The sections were air dried and fixed with 4% paraformaldehyde for 15min. After 3 washes in PBS, the slides were blocked with 5% milk in PBSfor 1 h, and then incubated overnight with anti-β₂-adrenergic receptorantibody (1:200, Santa Cruz Biotechnology) in blocking solution. Slideswere washed in PBS and incubated with secondary antibody (1:200, Cy3goat anti-rabbit, 16 h at 4° C.). Control slides were incubated withantibody specific blocking peptide to demonstrate specificity of bindingof the primary antibody. After washing with PBS, coverslips were mountedand viewed by epifluorescent microscopy.

As shown in FIG. 4, labeling with anti-β₂-adrenergic receptor antibodieswas considerably more intense in lungs from animals treated with nadololthan in lungs from untreated animals (A, control+antibody; B,control+antibody+blocking peptide; C, nadolol+antibody; D,nadolol+antibody+blocking peptide). Loss of this signaling uponincubation in the presence of the β₂-adrenergic receptor peptidedemonstrates that this antibody is specifically binding theβ₂-adrenergic receptor. This observation is consistent with theradioligand binding data of Example 2 and suggests that β₂-adrenergicreceptors are effectively upregulated by chronic administration ofβ₂-adrenergic inverse agonist drugs.

Example 3 Effect of Combination of Carvedilol and Salbutamol on AirwayHyperresponsiveness

The effect of combination therapy with carvedilol and salbutamol wascompared to monotherapy with carvedilol alone on airwayhyperresponsiveness in asthmatic mice.

Mice (Balb/cJ) aged 6 weeks were housed under specific pathogen-freeconditions and fed a chicken ovalbumin-free diet. Mice were systemicallysensitized with ovalbumin adsorbed to aluminum hydroxide. Mice weretreated as follows: CAR/SAL 28D=for 28 days mice (n=6-12) wereadministered carvedilol (2400 ppm in animal chow) and salbutamol(subcutaneous delivery of 0.5 mg/kg/day in an Alzet #2400 osmoticminipump); NTX S/C=mice (n=6-12) no drug treatment for 28 days;CTRL=mice (n=6-12) no drug treatment for 28 days, not subsequentlychallenged; CARHD 28D=for 28 days mice (n=6-12) were administeredcarvedilol only (2400 ppm in animal chow); CARHD 28D SAL AC=for 28 daysmice (n=6-12) were administered carvedilol (2400 ppm in animal chow) and15 minutes prior to measuring airway hyperresponsiveness, salbutamol wasadministered at a dose of 1.2 mg/kg.

To measure airway hyperresponsiveness after 28 days, all mice except theCTRL (control) mice were challenged with ovalbumin and then all micewere anesthetized, tracheotomized, and connected to a Flexivent smallanimal ventilator to measure airway resistance (R_(aw)) by the forcedoscillation technique. To induce airway constriction, a solutioncontaining 150 μg/mL of methacholine was infused using a syringeinfusion pump. The methacholine infusion was started at 0.008 mL/min andits rate was doubled stepwise up to a maximum 0.136 mL/min. Eachmethacholine dose was administered until a plateau was reached, duringwhich data were sampled at 1-min intervals for 3-5 min and thenaveraged.

In FIG. 5A, at the highest dose of methacholine, both of the combinationdrug treatments were equally effective in preventing bronchoconstrictionand not statistically significantly different from the control micewhich were only challenged with saline solution. The carvedilolmonotherapy resulted in higher bronchoconstriction than these treatmentsbut less than the non-drug treated sensitized and challenged (NTX S/C)mice. Thus, the combination therapy of β₂-adrenergic inverse agonist andagonist with the agonist administered either chronically or acutely iseffective at ameliorating airway hyperresponsiveness to allergen andmethacholine challenge and is an improvement over the monotherapy of theβ2-adrenergic inverse agonist alone.

This data is summarized in FIG. 5B, which shows that the combination ofcarvedilol and salbutamol is the most effective in reducing airwayhyperresponsiveness of the treatments for which the results are shown inFIG. 5A. This indicates the effectiveness of the use of combinationtherapy of β₂-adrenergic inverse agonist and agonist.

Example 4 Effect of Combination Therapy with Aminophylline on AcuteAirway Effects of Nadolol

Mice were sensitized to the allergen ovalbumin as described inExample 1. Mice were then challenged with allergen and then subjected tomethacholine-induced bronchoconstriction challenge, non-drug treated,NTX S/C, or pretreated with nadolol at 0.72 mg/kg i.p. for 15 minutesprior to methacholine challenge (nadolol acute treatment).

At time point 1 (time=−10 min) baseline airway resistance of the micewas determined. At time point 2 (time=−5 min) methacholine was infusedinto mice to reach their EC₇₀. At time point 3 (time=0 min)aminophylline was administered i.p. at a dose of 100 mg/kg.

In FIG. 6, pretreatment of mice with nadolol resulted in the samebaseline airway resistance as non-drug treated sensitized andallergen-challenged mice. However, upon methacholine challenge, thenadolol-treated mice exhibited a much higher airway resistance of ˜4.5versus 2.5 units. Upon administration of aminophylline, there was asignificant and sustained drop in airway resistance in both theuntreated and the nadolol-treated mice.

Z. Callaerts-Vegh et al., “Effects of Acute and Chronic Administrationof β-Adrenoceptor Ligands on Airway Function in a Murine Model ofAsthma,” Proc. Natl. Acad. Sci. USA 101: 49484953 (2004), have shownthat while nadolol administered chronically prevents airwayhyperresponsiveness in the same mouse asthma model, nadolol administeredacutely worsens airway hyperresponsiveness. These data demonstrate thatthe addition of the methylxanthine aminophylline can alleviate the acuteeffects on airway hyperresponsiveness of nadolol administration. This isbeneficial in that the opportunity exists for asthma subjects to takenadolol chronically to prevent bronchoconstriction. These subjects thencan co-administer a methylxanthine such as aminophylline to prevent theacute detrimental effects of nadolol.

Example 5 Effect of Treatment with Salbutamol or Nadolol on the Ratio ofPhospholipase C to Actin in Cultured Tracheal Smooth Muscle Cells

Cultured tracheal smooth muscle cells were obtained from mice exposed tothe following treatments: NS/NC=nonasthmatic, non-challenged mice;S/C=asthmatic mice; Sal.Ac=asthmatic mice, acute salbutamol treatment;Sal.Ch=asthmatic mice, chronic salbutamol treatment; Nad.Ac=asthmaticmice, acute nadolol high dose treatment; and Nad.Ch=asthmatic mice,chronic nadolol high dose treatment.

After airway function experiments, the trachea were surgically removedfrom anesthetized mice that had been treated with drugs or vehicle. Thetrachea was minced and the cells plated and grown in culture. The smoothmuscle cells grow faster and take over the culture dish. The cells weregrown in medium which contained the drugs used in the treatment orvehicle controls. Phospholipase C (PLC-β₁) was determined byimmunoblotting with an antibody specific for the enzyme. Actin was usedas a loading control and the amount of PLC-β₁ was expressed as a ratioto actin.

The phospholipase C protein level was measured in these cultured cellsand compared with the level of the structural protein actin as abaseline. The enzyme phospholipase C plays a key role in the pathwayleading to asthmatic symptoms, as it cleaves a phosphodiester bond inmembrane phospholipids, resulting in the formation of a 1,2-diglyceride.Arachidonate is then released from the diglyceride by the sequentialactions of diglyceride lipase and monoglyceride lipase. Once released, aportion of the arachidonate is metabolized rapidly, leading tooxygenated products, including eicosanoids such as prostaglandins. Thus,any treatment that can inhibit phospholipase C activity is relevant forthe treatment of asthma.

The results are shown in FIG. 7. The results shown in FIG. 7 indicatethat chronic administration of nadolol significantly decreases theactivity of phospholipase C. This indicates that such chronicadministration of nadolol is effective against asthma and preventsactivation of some of the mechanisms that lead to the symptoms ofasthma.

Example 6 Effect of β-Adrenergic Receptor Drugs at Low and High Doses onAirway Resistance

For these experiments, salbutamol was used for chronic administration at0.5 mg/kg/day with a minipump and for acute administration at 0.15 mg/kgby i.v. bolus 15 minutes prior to challenge. Alprenolol was used at ahigh dose of 7200 ppm in chow or at a low dose of 720 ppm in chow.Carvedilol was used at a high dose of 2400 ppm in chow or at a low doseof 720 ppm in chow. Nadolol was used at a high dose of 250 ppm in chowor at a low dose of 25 ppm in chow. Nadolol was also tested at 1 ppm inchow and these results were identical to the untreated mice.

The results are shown in FIGS. 8A (salbutamol); 8B (high-dosealprenolol); 8C (low-dose alprenolol); 8D (high-dose carvedilol); 8E(low-dose carvedilol); 8F (high-dose nadolol); and 8G (low-dosenadolol). In these diagrams, Ctrl=control mice, non-asthmatic, non-drugtreated; NTX=asthmatic mice, non-drug treated; AC=acute administration;2d=chronic administration for 2 days; 28d=chronic administration for 28days. The airway resistance (R_(aw)) is plotted as cm H₂O ml⁻¹ s. Thedata particularly shows the effect of the β-adrenergic inverse agonistscarvedilol and nadolol in providing protection from airwayhyperresponsiveness with chronic administration.

Example 7 Correlation of Decrease in Airway Resistance with Upregulationof β-Adrenergic Receptor Density

The correlation of the decrease in airway resistance with theupregulation of β-adrenergic receptor density for three differentperiods of administration of salbutamol, alprenolol, carvedilol, andnadolol is shown in Table 2. The periods of administration of the agentsare 15 minutes, 2 days, and 28 days. Only the inverse agonistscarvedilol and nadolol showed an increase in β-adrenergic receptordensity at periods longer than 15 minutes; carvedilol showed an increasein receptor density at 28 days, while nadolol showed an increase inreceptor density at both 2 days and 28 days. There was an exactcorrelation between the decrease of airway resistance (R_(aw)) and theincrease in receptor density. This strongly supports the concept ofcombination therapy, such as with an inverse agonist and an agonist.

TABLE 2 Correlation of Decrease In Airway Resistance With Upregulationof β₂-Adrenergic Receptor Density 15 minutes 2 days 28 days IncreasedIncreased Increased Decreased R_(aw) β₂AR density Decreased R_(aw) β₂ARdensity Decreased R_(aw) β₂AR density Salbutamol yes yes no no no noAlprenolol yes yes no no no no Carvedilol no no no no yes yes Nadolol nono yes yes yes yes

Example 8 Effects of Chronic Treatment with Metoprolol and Timolol onAirway Hyperresponsiveness in Asthmatic Mice

The protocols of Example 1 were followed for two additional inverseagonists, metoprolol (dosage of 20 mg/kg administered 3× daily viasubcutaneous injection for 7 days) and timolol (dosage of 20 mg/kg inchow for 7 days), using asthmatic mice and methacholine challenge as inExample 1. Airway resistance (R_(aw)) was measured as in Example 1. Theresults for metoprolol and timolol are shown in FIG. 9A. The resultswere compared to historical controls as shown in FIG. 9B: Ctrl, no drugtreatment, no challenge with methacholine; NTX, no drug treatment,challenged with methacholine. The results indicate that chronictreatment with both metoprolol and timolol are effective in reducingairway hyperresponsiveness in asthmatic mice.

ADVANTAGES OF THE INVENTION

The present invention provides a improved method of treating chronicpulmonary airway diseases such as asthma, emphysema, and chronicobstructive pulmonary diseases and avoids the tolerance or tachyphylaxisthat often is the consequence of conventional therapy with β-adrenergicagonists. The use of inverse agonists, in essence, forces the body torespond by improving its own signaling mechanisms to counter thepulmonary airway disease. Accordingly, compositions and methods thatemploy inverse agonists have broad potential for treating such diseasesand conditions without the induction of tolerance. This promisessuperior long-term results in the treatment of such conditions withoutinterfering with short-term acute therapy.

The inventions illustratively described herein can suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising,” “including,” “containing,” etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the future shown and described or anyportion thereof, and it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions herein disclosed can be resorted bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of the inventions disclosed herein.The inventions have been described broadly and generically herein. Eachof the narrower species and subgeneric groupings falling within thescope of the generic disclosure also form part of these inventions. Thisincludes the generic description of each invention with a proviso ornegative limitation removing any subject matter from the genus,regardless of whether or not the excised materials specifically residedtherein.

In addition, where features or aspects of an invention are described interms of the Markush group, those schooled in the art will recognizethat the invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. It is also to beunderstood that the above description is intended to be illustrative andnot restrictive. Many embodiments will be apparent to those of in theart upon reviewing the above description. The scope of the inventionshould therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to theappended claims, along with the full scope of equivalents to which suchclaims are entitled. The disclosures of all articles and references,including patent publications, are incorporated herein by reference.

I claim:
 1. A method for treatment of pulmonary airway disease in asubject suffering from pulmonary airway disease comprising chronicallyadministering a therapeutically effective amount of a β-adrenergicinverse agonist to the subject to treat the pulmonary airway disease byaction of inverse agonism to promote long-term upregulation of thepopulation of β-adrenergic receptors upon chronic administration of theβ-adrenergic inverse agonist, wherein the β-adrenergic inverse agonistexhibits inverse agonist activity at the β₂ receptor of bronchialtissue, wherein the β-adrenergic inverse agonist is selected from thegroup consisting of nadolol, bupranolol, butoxamine, carazolol,ICI-118,551, levobunolol, metoprolol, and timolol, and wherein suchchronic administration requires increasing dosages over time.
 2. Themethod of claim 1 wherein the β-adrenergic inverse agonist is nadolol.3. The method of claim 1 wherein the β-adrenergic inverse agonist isadministered by a route selected from the group consisting of oral,sustained-release oral, parenteral, sublingual, buccal, insufflation,and inhalation.
 4. The method of claim 3 wherein the β-adrenergicinverse agonist is administered by the sustained-release oral route. 5.The method of claim 1 wherein the pulmonary airway disease is selectedfrom the group consisting of asthma, bronchiectasis, bronchitis, chronicobstructive pulmonary disease, Churg-Strauss syndrome, pulmonarysequelae of cystic fibrosis, emphysema, allergic rhinitis, andpneumonia.
 6. The method of claim 5 wherein the pulmonary airway diseaseis asthma.
 7. The method of claim 5 wherein the pulmonary airway diseaseis chronic obstructive pulmonary disease.
 8. The method of claim 5wherein the pulmonary airway disease is emphysema.
 9. The method ofclaim 1 wherein the β-adrenergic inverse agonist is administered overtime in a series of graduated doses starting with the lowest dose andincreasing to the highest dose, wherein such graduated doses aredetermined by determination of the increase of receptor density.
 10. Themethod of claim 9 wherein, when the highest dose is reached, theβ-adrenergic inverse agonist continues to be administered at that dose.11. The method of claim 1 further comprising administering atherapeutically effective amount of an additional agent selected fromthe group consisting of: a β₂-selective adrenergic agonist; a steroid;an anticholinergic drug; a xanthine compound; an anti-IgE antibody; aleukotriene modifier; and a phosphodiesterase inhibitor in order totreat the pulmonary airway disease by action of inverse agonism topromote long-term upregulation of the population of β-adrenergicreceptors upon chronic administration of the β-adrenergic inverseagonist, wherein the β-adrenergic inverse agonist exhibits inverseactivity at the β₂ receptor of bronchial tissue.
 12. The method of claim11 wherein the additional agent is a β₂-selective adrenergic agonist andthe β₂-selective adrenergic agonist is selected from the groupconsisting of albuterol, bitolterol, clenbuterol, clorprenaline,dobutamine, fenoterol, formoterol, isoetharine, isoprenaline,levabuterol, mabuterol, metaproterenol, pirbuterol, ritodrine,salbutamol, salmeterol, terbutaline, and the salts thereof.
 13. Themethod of claim 11 wherein the additional agent is a steroid and whereinthe steroid is selected from the group consisting of beclomethasone,budenoside, ciclesonide, flunisolide, fluticasone, methylprednisolone,prednisolone, prednisone, and triamcinolone, and the salts thereof. 14.The method of claim 11 wherein the additional agent is ananticholinergic drug and wherein the anticholinergic drug is selectedfrom the group consisting of ipratropium bromide, tiotropium bromide,and oxitropium bromide, and the salts thereof.
 15. The method of claim11 wherein the additional agent is a xanthine compound and wherein thexanthine compound is selected from the group consisting of theophylline,extended-release theophylline, aminophylline, theobromine, enprofylline,diprophylline, isbufylline, choline theophyllinate, albifylline,arofylline, bamifylline and caffeine.
 16. The method of claim 11 whereinthe additional agent is an anti-IgE antibody and wherein the anti-IgEantibody is a monoclonal antibody or a genetically engineered antibodythat is derived from a monoclonal antibody.
 17. The method of claim 11wherein the additional agent is a leukotriene modifier and wherein theleukotriene modifier is selected from the group consisting of ibudilast,montelukast, pranlukast, and zafirlukast, and the salts thereof.
 18. Themethod of claim 11 wherein the additional agent is a phosphodiesteraseIV inhibitor and wherein the phosphodiesterase IV inhibitor is selectedfrom the group consisting of roflumilast and cilomilast, and the saltsthereof.